IN-VITRO PHARMACOLOGICAL EVALUATION OF FRESH LEAF DECOCTION OF

CARICA PAPAYA L.

A Project Submitted to
JAWAHARLAL NEHRU TECHNOLOGICAL UNIVERSITY ANANTAPUR
In partial fulfilment for the award of Degree of

MASTER OF PHARMACY
By
Mr.(S0102)

Under the Guidance of

Prof. M .Pharm.,Ph.D.,M.Sc.,

)-515001, A.P ,INDIA

1
CONTENTS :
• INTRODUCTION AND RATIONALE
• LITERATURE REVIEW
• PLANT PROFILE
• AIM AND OBJECTIVES
• MATERIALS AND METHODS
• EXPERIMENTAL INVESTIGATIONS
• DISCUSSION OF RESULTS
• SUMMARY AND CONCLUSION
• FUTURE SCOPE
• REFERENCES

2
 INTRODUCTION AND RATIONALE :
• Increased adoption of maladaptive diet is compromising the quality of
human life.
• Complementary and alternative therapies are fruitful in disease
prevention and treatment.
• Dietary disease management is the current trend which is as per
Hippocrates statement of “Let food be your medicine and medicine be
your food”.
• Hence ethical investigation of natural substances as per traditional
drug development paradigm to bring the pipeline dream of dietary
disease management.
• Current nCovid-19 management protocols are prioritizing the concept
of dietary disease management.

3
Biological screening methods:
 In-vitro methods

 In-vivo methods
 Ex-vivo methods
 In- silico methods

4
 In-vitro screening assays –pros & cons
 screening, relatively inexpensive, easy to set up, quick yield of results. 1
 Alternative to animal screening models in accordance with the objective
of ICH 3Rs- Reduction, Replacement and Refinement of procedures
involving animals and free from ethical committee approvals. 2

 Elucidation of cellular or molecular working mechanisms can be

achieved.

 Cells are treated outside the physiological environment and are ideal to
measure a specific dynamic effect like pharmacological, biochemical or
molecular, immunological, genetic, physiological or pathological.

5
• Red blood cells (RBCs) are one of the most susceptible biological tissues to
oxidative stress due to the presence of both high concentration of
polyunsaturated fatty acids (PUFA) in the membrane and the oxygen transport
associated with redox active hemoglobin molecules, which are promoters of
reactive oxygen species (ROS).
• The occurrence of lipid peroxidation due to any reason causes alteration in RBC
membrane structure, function and alteration of membrane bound receptors and
enzymes.
• No earlier reports were found regarding the in –vitro anti-oxidant activity of
fresh leaf decoction of Carica papaya L. with respect to the catalytic potential
of RBC membrane bound enzymes and cytosolic metabolic enzymes against
Phenylhydrzine induced goat erythrocyte membrane damage.Hence the present
study was undertaken to evaluate the potential of leaf decoction of Carica
papaya L. for its antioxidant an anti-hemolytic activity by in-vitro
pharmacological screening methods .

6
REVIEW OF LITERATURE
S.No. Earlier research carried on YEAR JOURNAL REFERENCE
Carica papaya L.

1 A review of pharmacognostic, 2017 Journal of Scientific Hafiz Abdul Khaliq

physicochemical, phytochemical and and Industrial
pharmacological studies on Carica papaya3 Research(JSIR)

2 Evaluation of phytochemical constituents and 2016 Journal of Global Lamia abdul majeed
anti-oxidant status for Carica papaya4 Trends in Almashhedy, Hawraa
Pharmaceutical S Al-Kawaz
Sciences

3 Determination of flavoniods in papaya fruit 2016 The scientific world Tripathi et al.,,
and effects of flavonoids on blood glucose journal
level5

4 Physicochemical and antioxidant properties 2015 Biological and Ramesh Singh Pal,
of papaya fruit as a function of cultivar and applied sciences et al.,
fruit harvested month.6

8
S.N Earlier research YEAR JOURNAL REFERENCE
O
carried on Carica
papaya L.

5 Papaya fruit allevatives learning 2017 Oxidative Medicine and Wei-Zhen Xue
and memory deficits induced by Cellular Longevity ush ,et al.,
Pb through antioxidation :In -
vitro & In- vivo studies15

6 The papaya fruit peptide 2014 The Journal Of C Ciacci, et al .,

displays anti-inflammatory and Translational Immunology
anti-oxidant effects in in-vitro &
Ex-vivo in human intestinal
models16

7 Determination of the effect of 2013 The Pharma Innovation Dr.Elaine Rush , et

lyophilised papaya fruit on Journal al .,
digestion of protein : In vitro &
In vivo17

8 Putative mechanisms of Papaya 2018 Critical reviews in food Simone Birgit

fruit on maintenance of normal science and nutrition Bayer, et al.,
Gastro Intestinal function 18
9
S.No. Earlier research YEAR JOURNAL REFERENCE
carried on Carica
papaya L.

9 Phytochemical analysis 2018 Journal of Biomedical Sadia Rafique.,

and anti-oxidant activity research et al.,
of Pesia americana and
Carica papaya fruit
extracts by DPPH
method19
10 Nutritional and health 2018 European journal of David.P.Richar
attributes of papaya leaf nutrition dson., et al.,

11 Nutritional content and 2018 World journal of Deepak Kumar

therapeutic potential of pharmacy and Puri.,et al.,
papaya fruit pharmaceutical
sciences

12 Characterization of 2017 The pharma innovation Rehana Salim.,

chemical and anti- journal et al.,
oxidant properties of
10
papaya fruit22
Review on Na+/ K+ ATPase Assay:
S.NO TITLE OF THE YEAR JOURNAL REFERENCE
WORK

1 Aqueous bark extract of 2018 International Journal of Pharm SUDESHNA PAUL.,

Terminalia arjuna protects Pharm Sciences et al.,
against PHZ induced
oxidative damage in goat
RBC cell membrane bound
and metabolic enzymes.7

2 A quick assay for NA+-K+- 2002 Journal of Global Trends in Pradip Sarkar
ATPase specific activity8 Pharmaceutical Sciences

11
S.NO RESEARCH WORK YEAR JOURNAL REFERENCE
CARRIED on Na+/ K+
ATPase Assay

3 In Vitro pharmacological 2013 The Pharma innovation Rozina Parul,

Activity Of Methanol Extracts journal Sukalayan Kumar
Of Three Bangladeshi Kundu , and Pijush
Medicinal Plants and their Saha
products11

4 Antioxidant activity of different 2012 Asian specific journal of Badakhshan Mahdi -

parts of kiwi fruit12 tropical biomedicine. Pour

12
Review on Acetylcholinesterase activity:
S.NO Review on YEAR JOURNAL REFERENCE
Acetylcholinesterase
activity

1 Cholinesterase inhibitors modify 2004 World Journal of Darvesh, Sultan et al.,

the activity of intrinsic cardiac Pharmacy and
neurons9 Pharmaceutical
Sciences

2 A new and rapid colorimetric 1961 Journal of George L.Ellman, et al.,

determination of Scientific &
acetylcholinesterase activity 10 Innovative
Research

13
 Review on Hexokinase assay:
S.NO RESEARCH WORK YEAR JOURNAL REFERENCE
CARRIED on Na+/ K+
ATPase Assay

1 In vitro inhibition of human 2017 Journal of biochemical and Jalal A.

erythrocyte membrane bound molecular toxicology Aljamal,Muwaffag
enzymes by various drugs13 Badawneh

2 Rabbit red blood cell ATPase 1984 Molecular and Cellular Mauro Magnani, et
assay14 Biochemistry al.,

14
 Review on in-vitro NO radical scavenging assay:
S.NO RESEARCH WORK YEAR JOURNAL REFERENCE
CARRIED on Na+/ K+
ATPase Assay

1 In vitro free radical 2012 Journal of biochemical Dharmendra singh

scavenging activity of and molecular et al.,
Scintella asciatica 13 toxicology

2 Regulatory effect of three medicinal plants 2013 Molecular and Cellular Rozina parul et
on nitric oxide (NO) levels using sodium toxicology al.,
nitroprusside as a NO donor in- vitro.14

15
 Review on in-vitro Nitric Oxide radical scavenging assay:
S.NO RESEARCH WORK YEAR JOURNAL REFERENCE
CARRIED on Na+/ K+
ATPase Assay

3 Antioxidant potential of 2012 Journal of Sridhar Prasad Y

aqueous extract of S.indicum ethnobiology. et al,.
L. supplementation therapy in
a rodent model of hemolytic
anemia induced by
phenylhydrazine (PHZ)

4 The anti-anemia activity of 2014 Journal of Radiation Hye Won Lee et

the H2O extracts from Biology. al,.
constituent herbal medicines
of Samul-tang in an anemia
model induced by
intravenous infection of
phenylhydrazine-HCL (PHZ)

16
PLANT PROFILE OF CARICA PAPAYA L.

CARICA PAPAYA L PLANT.FOLIAGE

17
 Chemical constituents present in Carica papaya L.

• The leaves of Carica papaya L. contain the alkaloid carpaine 24,

pseudocarpaine25 and dehydrocarpaine I & II.
• Contains high levels of vitamin E and vitamin C, sertonin and
potassium.

• The alkaloid Carpaine has been found to possess antitumor

activity in-vitro against mouse lymphoid leukemia .
• Seven flavonoids including quercetin 3-(2G-rhamnosyl-
rutinoside), kaempferol 3-(2G-rhamno-syl-rutinoside),
quercetin 3-rutinoside, myricetin 3-rhamnoside, kaempferol 3-
rutinoside, quercetin, and kaempferol have also been reported
and has been assessed for their antioxidant activities
18
 Claimed medicinal uses

• Leaves:
• Papaya leaf has a numberless of benefits and in some parts of Asia, the young leaves of the papaya are
steamed and eaten like spinach.
• a. Dengue fever management: Dr. Sanath Hettige, who conducted the research on 70 dengue fever
patients, said papaya leaf decoction helps increase white blood cells and platelets, normalizes ,clotting,
and repairs the liver.
• b. Cancer Cell Growth Inhibition: Recent research on Papaya leaf tea extract has demonstrated cancer cell
growth inhibition. It appears to boost the production of key signalling molecules called Th1-type
cytokines, which help regulate the immune system.
• c. Antimalarial and anti plasmodial activity
• Antimalarial and anti plasmodial activity has been noted in some preparations of the plant, but the
mechanism is not understood and not scientifically proven
• d. Facilitation of digestion:

• Fruit
• Papaya fruit is a rich source of nutrients such as pro vitamins, carotenoids, vitamin C, B lycopene, dietary minerals and dietary fibre.
Danielone is a phytoalexin found in the papaya fruit is powerful antioxidant..
• Laxative: Ripe papaya fruit is laxative which assures of regular bowel movement.The milky decoction which is tapped from the
green, mature fruit while still in the tree contains an enzyme known as "papain". People use this in the preparation of different
remedies for indigestion

19
AIM AND OBJECTIVES
• The aim of our present study was to carry out in- vitro pharmacological screening of
fresh leaf decoction of Carica papaya against phenylhydrazine induced RBC membrane
damage for its anti-oxidant and anti-hemolytic activity.
OBJECTIVES
• The objectives of the study include the following .
• 1.Preparation of fresh leaf decoction of Carica papaya.
• 2 Estimation of percentage yield of decoction.
• 3.Random determination of weight/volume of decoction.
• 4. Determination of protein concentration in LDCP
• by Bradford method.
• Evaluation of the LDCP for various In vitro Pharmacological activities with respect to
erythrocyte membrane bound cum metabolic enzyme activity assays.
A. In-vitro Na-K+ ATPase assay
B. In-vitro Acetylcholinesterase activity
C. Nitric oxide Radical Scavenging Activity
D. Hexokinase assay.

20
 MATERIALS AND METHODS

1) Preparation of Carica papaya L leaf decoction: Freshly collected leaves

of Carica papaya L are washed thoroughly to remove adherent impurities,shade dried .
100 gm of dried leaves were boiled in 100 ml of water with a pinch of salt for 20
minutes.The obtained decoction is strained through muslin cloth and is preserved for
further processing. Volume of the obtained decoction was measured with a measuring
cylinder to calculate the percentage yield .

21
2) Random determination of wt/ml
• 1ml of fresh decoction was transferred into a previously weighed
clean specific gravity bottle and the weight was noted as W 1, placed
in hot air oven at a temperature of around 40-450c until a semisolid
consistency obtained. Its weight was noted as W2.
• The difference in weights i.e W1 - W2 gives the weight/volume(ml)
of the decoction.
• The value obtained is employed for the preparation of suitable
dilutions of the decoction for evaluation of further parameters.
% yield of fruit decoction = volume of decoction obtained in ml ×
100
wt of fruit slices in grams
% yield of fruit decoction = 62 × 100 =51.6 %
120
22
3)Preparation of 20μg/ml ,40 μg/ml ,60 μg/ml, 80
μg/ml and 100μg/ml concentrations of LDCP39:
• The wt/ml value of LDCP was employed to prepare the
intended dilutions of the sample by using the formula:
C1V1 = C2V2 where
C1= concentration of stock solution
C2= Desired concentration of sample solution to be used in
the assay
V1= volume of stock solution needed to make the solution of
desired concentration
V2= Volume of drug solution to be employed in the assay .

23
4)Estimation of total protein concentration in LDCP (bradford method)40
The basis of this assay is absorbance maximum of an acidic solution of Coomassie
Brilliant
• REAGENTS & CHEMICALS

1.Bovine Serum Albumin (1 mg/ml)

2.Bradford reagent

3.Fresh leaf decoction of Carica papaya sample ( LDCP)

Protein concentration in LDCP(ug / ml )= Absorbance х Dilution factor /slope(1/

(μg)× volume of diluted protein used for the assay(ml)

Blue G- 250 shifts from 465nm to 595nm upon protein binding

24
4) In-vitro Na+/K+ ATP-ase assay (Svoboda and Mosinger ,1981)41
Step 1:Processing of goat blood to obtain whole RBCs 12
Collection of Goat blood (from
Municipal Corporation approved slaughter house) into heparin coated vacutainer

Centrifuge at 3000rpm for 10 minutes at 4 0C using cooling centrifuge

Removal of Plasma and the buffy coat by aspiration

Collection of whole RBCs

25
Wash the collected whole RBCs thrice with 0.9% NaCl
Step 2:Incubation of whole RBCs with Phenylhydrazine (to serve
as Group II)
500 μl of the prepared whole RBC suspension
+

500 μl of 1mM Phenylhydrazine prepared in 50 mM sodium phosphate buffer (pH

7.4)

Incubate at 370C in a shaking water bath for 1 hour

Terminate the incubation by addition of 100μl of 16mM EDTA prepared in 50 mM

sodium phosphate buffer (pH 7.4)

Wash the PHZ treated whole RBCs thrice with 0.9% NaCl and subject for hypotonic
lysis

26
 Step 3: Grouping of whole RBCs for further treatment: The
whole RBCs were divided into SEVEN groups .
• Group I: Normal Control : This group contains the untreated RBC
preserved in Sodium phosphate buffer to measure the activity of
Na+/K+ATPase and the liberation of inorganic phosphate.
• Group II: Phenyl hydrazine treated can serve as negative control. The
purpose of this group is to determine the extent of damage induced by
Phenyl hydrazine to the RBC membrane and the membrane bound
Na+/K+ATPase.
• Groups III: PHZ treated + LDCP at a concentration of 20µg/mL.
• Group IV: PHZ treated + LDCP at a concentration of 40µg/mL.
• Group V: PHZ treated + LDCP at a concentration of 60µg/mL.
• Group VI: PHZ treated +LDCP at a concentration of 80µg/mL.
• Group VII: PHZ treated + LDCP at a concentration of 100µg/mL.
• Group III to Group VII were employed to evaluate the protective effect of
LDCP on Phenyl hydrazine induced hemolysis.

27
STEP 3 :Incubation of whole RBCs with LDCP at various concentrations and
Phenylhydrazine (Groups III to VII)

500 μl of the prepared whole RBC suspension

+
500 μl of LDCP (20,40.60,80 and 100 µg/mL)
+
500 μl of 1mM Phenylhydrazine prepared in 50 mM sodium phosphate buffer (pH 7.4)

Incubate at 370C in a shaking water bath for 1 hour

Terminate the incubation by the addition of 100μl of 16mM EDTA prepared in 50 mM

sodium phosphate buffer (pH 7.4)

Wash the PHZ treated whole RBCs thrice with 0.9% NaCl and subject for hypotonic
lysis

28
Step IV: Preparation of erythrocyte membrane of the
treated and untreated groups of RBCs
• Haemoglobin-free erythrocyte membrane (either normal or
treated) was prepared according to method of Arduini et al. The
washed erythrocytes (100µl) were subjected to hypotonic lysis in
40 volumes of 5 mM sodium phosphate buffer (pH 8.0) and
centrifuged at 4,000 rpm for 20 min at 4°C.
• The supernatant, thus obtained, was discarded and pellet was
washed at least three times in the same buffer until a colorless
pellet was obtained.
• The erythrocyte ghosts were suspended in the same buffer and
stored at –20°C for future use.

29
 Determination of erythrocyte membrane ‑ bound enzyme
Na+ K+ ATPase activity42:
(Method of Svoboda and Mosinger)
• 250 µl of 10mM tris HCl buffer followed by the addition of 50 µl
of 60 mM NaCl, 50 µl of 50 mM KCl, along with 50 µl of 1 mM
Na. EDTA, and 50 µl of 80 mM ATP. The reaction mixture was
preincubated at 37°C for 10 min. Then, 25 µl of 10% homogenate
was added to the test alone and further incubated at 37°C for 1h.
The reaction was immediately arrested by the addition of 10%
TCA.
• The control reaction rate was correspondingly assessed by adding
25 µl of 10% homogenate only after arresting the reaction. The
precipitate was removed by centrifugation at 3500 rpm for 10 min.

30
 Step V: Estimation of inorganic phosphate from the protein
free filtrate (Fiske and Subbarow ,1925)43
Assay system for estimation of inorganic phosphate

1.Molybdate Solution: 25 gm of reagent grade ammonium molybdate was dissolved in 200m! Of water.
In one liter volumetric flask, 300ml of 1O N sulphuric acid was taken. Molybdate solution was added
to this and the volume was made up to one liter.

2.Aminonaphthosulphonic acid reagent (ANSA reagent): 0.5 gm of 1,2,4, amino naphthosulphonic

acid was added to 195 ml of 15% sodium bisulphite solution ·and shaken till the powder dissolved.
5ml of 20% sodium sulphite solution was added to it. Solution was preserved in a dark bottle in cold.

3. Trichloroacetic acid (10%) - Dissolve 100 g of high grade crystalline acid in distilled water and dilute
to 1 Liter.

4. 10 N Sulphuric acid - Slowly add 450 ml of concentrated sulphuric acid to 1300 ml distilled water in a
pyrex container. 4. 15% sodium bisulphite solution - Add 30 g of reagent grade sodium bisulphate in
distilled water and dilute to 200 ml. Clear supernatant was employed.

5. 20% sodium sulphite solution - Dissolve 20 g of reagent grade anhydrous sodium sulphite in
distilled water and dilute to 100 ml.

31
• Estimation of Inorganic phosphate: 1ml of the protein-free
supernatant was taken, to which 1 ml of distilled water was
added. 0.5ml of ammonium molybdate solution was added and
incubated for 10 min at room temperature. 0.4 ml of ANSA
reagent was added and the blue colour developed was estimated
spectro photometrically at 640nm. The amount of inorganic
phosphate liberated micrograms of inorganic phosphate released
per mg protein per minute from the standard curve of inorganic
phosphate obtained by using KH2PO4. 15
• Amount of inorganic phosphate released per mg protein per
minute = Amount of pi of Control-Amount of pi of Test

32
6) IN-VITRO ASSAY OF RBC MEMBRANE BOUND ACETYLCHOLINESTERASE
ACTIVITY:

2.PRINCIPLE:
Ellman Esterase Assay:
• AchE hydrolyses the acetylthiocholine to produce thiocholine and
acetate. The thiocholine in turn reduces the Dithiobis-
Nitrobenzoic Acid liberating nitrobenzoate. The assay is based on
measurement of the change In absorbance at 405 nm.

Acetylthiocholine Acetylcholinesterase
T hiocholine + Acetate

Thiocholine + 5,5-dithiobis-2-nitrobenzoate 5-thio-2-

nitro-benzoicacid

33
3. PREPARATION OF REAGENTS:2
1. 0.1M Phosphate buffer:
 Solution A:5.22g of K2HPO4 and 4.68g of NaH2PO4 are
dissolved in 150 ml of distilled water
 Solution B:6.2g NaOH is dissolved in 150ml of distilled water
 Solution B is added to solution A to get the desired pH (pH8.0
or 7.0) and then finally the volume is made up to 300 ml with
distilled water.
2. 5,5’-dithiobis-(2-nitrobenzoic acid) (DTNB):
39.6mg of DNTB with 15mg NaHCO3 is dissolved in10mlof0.1M
phosphate buffer.
3. Acetyl thio choline iodide:
21.67mg of Acetyl thio choline iodide is dissolved in 1ml of water.
4. RBC Suspension (Test System):
The 100 μl of blood cells were suspended in 60ml of buffer. 34
• The Enzyme activity, R was calculated by using the
formula
Enzyme activity, R= 5.74× 10× A
CO

• Where., R= Rate in moles of substrate hydrolyzed/minute/gm

A=Change in absorbance/min
CO=Original concentration (mg/ml)

35
 NITRIC OXIDE RADICAL SCAVENGING
ACTIVITY:43
1. Collection of RBC Pellets:
The blood was collected from the Local Laboratory, the blood was centrifuged with 0.9% Nacl
for 3 times, the obtained RBC pellets were stored in the refrigerator for the activities.
2. Reagents:
a) preparation of sodium phosphate buffer – 0.272g of Na 2HPO4 and 1.1g of NaH2PO4 in
200ml of water, maintained the p H 7.4.
b) 10mM Sodium nitroprusside
c) Griess reagent – 0.5g of griess reagent was dissolved in 10ml of distilled water
d) Preparation of Groups and concentrations of the kiwi fruit decoction
Groups – Group 1 – RBC
Group 2 – RBC + PHZ
Group 3 – RBC + 20ug/ml conc. of LDCP + PHZ
Group 4 – RBC + 40ug/ml conc. of LDCP conc. of decoction + PHZ
Group 5 – RBC + 60ug/ml conc. of LDCP conc. of decoction + PHZ
Group 6 – RBC + 80ug/ml conc. of LDCP conc. of decoction + PHZ
Group 7 – RBC + 100ug/ml conc. of LDCP conc. of decoction + PHZ
36
Assay Procedure For NO Radical Scavenging Activity:

3ml of 10mM of sodium nitroprusside solution in phosphate

buffer saline and 1ml of different concentrations of leaf decoction

Incubated for 3 hours at room temperature and 2ml of griess

reagent was added later.

Incubated for 30min and absorbance was measured at 540 nm

using Visible spectrophotometer.
37
 HEXOKINASE ASSAY:

Hexokinase D was assayed by the method of Brandstrup et

al., (1957).
Reagents
1. 0.05 M Glucose solution
2. 0.72 M ATP solution
3. 0.05 M Magnesium chloride (MgCl2)
4. 0.0125 M Di potassium hydrogen phosphate solution
5. 0.1 M Potassium chloride solution
6. 0.5 M Sodium fluoride solution
7. 0.01 M Tris-HCl buffer, pH 8.0
38
1ml of glucose solution, 0.5ml of magnesium chloride solution, 0.5ml
of dipotassium hydrogen phosphate, 0.4ml of potassium chloride,
0.1ml of NaF and 2.5ml of tris HCl buffer was taken into test tube.

mixture was pre incubated at 370c for 15mins.

Add 1.0 ml of fruit decoction (different concentrations)

Aliquot was taken after 30 min of incubation at 37 C

•

39
Subjected for centrifugation, glucose in the
supernatant was estimated by the O-toluidine
method of Sasaki and Matsui

A reagent blank was run with each test. The

difference between the two values gave the
amount of glucose phosphorylated.

40
EXPERIMENTAL INVESTIGATIONS

41
 Estimation of Total protein present in LDCP
Optical density (595nm)readings of varied amount (μg) of Bovine serum
albumin (Protein standard)

42
Plot of Absorbance at 595 nm against the amount of
Bovine serum albumin (μg)

43
Absorbance (595nm) values of different dilutions of Test
(LDCP) sample
S. No. Volume of leaf decoction of Carica Absorbance(595 nm)
papaya L (μl)

1 100 (1:1000 dilutions) 0.007

2 100 (1:100 dilutions) 0.028

3 100 (1:10 dilutions) 0.426

4 100 (undiluted) 1.278

Protein concentration=Absorbance × Dilution

factor/slope(1/µg)×volume of the diluted protein used for the assay(ml)
Protein concentration in
LDCP=0.426×10/0.004×0.1=10650µg/ml=10.56mg/ml

44
Effect of LDCP treatment on Na+/K+ ATPase activity in liberating inorganic
phosphate (Pi)

S. No. Group Amount of Pi (µg of Pi liberated/min/mg

of protein)

1 Normal Control 60.247±0.061

2 PHZ Treated 10.745±0.495
3 LDCP Treated (20 µg/ml) 23.117±0.536****
4 LDCP Treated (40 µg/ml) 31.370±0.494 ****
5 LDCP Treated (60 µg/ml) 38.467±0.371****
6 LDCP Treated (80 µg/ml) 48.600±0.775****
7 LDCP Treated (100 µg/ml) 58.153±0.654****

The values expressed were the Mean ± SEM of 3 observations (n=3)

**** indicates significant when compared with PHZ treated ., (p<0.0001)

45
Effect of fresh leaf decoction of Carica papaya L.Actindia on RBC membrane bound
Na+/K+ ATPase

g of Pi liberated/ min/mg of protein

80

60
LDCP Treated (80 µg/ml)
LDCP Treated (60 µg/ml)
40
LDCP Treated (40 µg/ml)
LDCP Treated (20 µg/ml)
20
LDCP
Treated
0 (100µg/ml)
PHZ TREATED
CONTROL

TREATMENT GROUPS

46
Effect of LDCP treatment on Acetylcholinesterase activity in treated Goat RBCs

S. No. Group Acetylcholinesterase activity

(Units/min/mg of protein)
1 Normal Control 66.767±1.271
2 PHZ Treated 23.610±0.370
3 LDCP Treated (20 µg/ml) 26.27±0.615ns
4 LDCPP Treated (40 µg/ml) 34.153±0.483 ns

5 LDCPTreated (60 µg/ml) 41.473±0.596****

6 LDCP Treated (80 µg/ml) 53.40811±0.918****
7 LDCP Treated (100 µg/ml) 60.513±0.376****

• Effect of LDCP treatment on Acetylcholinesterase activity .

The values expressed were the Mean ± SEM of 3 observations (n=3)
**** indicates significant when compared with PHZ treated ., (p<0.0001)

47
Acetylcholinesterase activity in LDCP treated Goat RBC membranes
80
CONTROL

Units/min/mg of protein
PHZ treated
60
LDCP treated (20g/ml)
40 LDCP treated (40µg/ml)
LDCP treated (60µg/ml)
20 LDCP treated (80µg/ml)
LDCP treated (100µg/ml)
0

TREATMENT GROUPS

48
Effect of leaf decoction of Carica papaya L on nitric oxide
concentration in Goat RBCs

S. No. Group NO concentration (µmoles of nitrate/mg of

protein)

1 Normal Control 6.210±0.184

2 PHZ Treated 18.470±0.447

3 LDCP Treated (20 µg/ml) 17.943±0.509 ns

4 LDCP Treated (40µg/ml) 15.853±0.302 ns

5 LDCP Treated (60 µg/ml) 14.353±0.262 ns

6 LDCP Treated (80 µg/ml) 12.597±0.697****

7 LDCP Treated (100 µg/ml) 10.267±0.378****

49
Nitric oxide concentration in LDCP treated Goat RBC

20
CONTROL

PHZ treated

15
LDCP treated (20 µg/ml)
m of nitrate/mg of protein

LDCP treated (40 µg/ml)

10
LDCP treated (60 µg/ml)

LDCP treated (80 µg/ml)

5
LDCP treated (100 µg/ml)

0
L d l) l) l) l) l)
O ate /m /m /m / m /m
R
N
T tr
e µg µg µg µg µg
O Z 2 0 4 0 60 8 0 0 0
C ( ( ( ( (1
PH ed ed ted ted ed
t t
ea trea trea rea eat
tr t tr
P P P P P
C DC DC C C
LD L L LD LD
Treated groups

50
 Effect of LDCP treatment on Hexokinase activity in
treated Goat RBCs

S. No. Group Hexokinase activity (Units/min/mg of protein)

1 Normal Control 5.480±0.315
2 PHZ Treated 1.033±0.109
3 LDCP Treated (20 µg/ml) 1.337±0.066 ns
4 LDCP Treated (40 µg/ml) 1.440±0.053 ns
5 LDCP Treated (60 µg/ml) 1.900±0.072 ns
6 LDCP Treated (80 µg/ml) 1.840±0.062 ns
7 LDCP Treated (100 µg/ml) 2.503±0.093****
Table No.5.7. Effect of LDCP treatment on Hexokinase activity .
The values expressed were the Mean ± SEM of 3 observations (n=3)
**** indicates significant when compared with PHZ treated ., (p<0.0001)

51
Units/min/mg of protein Hexokinase activity in LDCP treated Goat RBCs

8
Control
PHZ treated
6 LDCP treated (20 µg/ml)
LDCP treated (40 µg/ml)
LDCP treated (60 µg/ml)
4
LDCP treated (80 µg/ml)
LDCP treated (100 µg/ml)
2

TREATMENT GROUPS

52
DISCUSSION OF RESULTS:
• Mitochondrion generates more number of free radicals in the
process of electron transport chain in cell.
• Free radicals have both physiological and pathological roles.
• Oxidative stress develops due to imbalance between pro and
anti oxidant status
• All biological molecules are prone to free radical attack.

53
• Red blood cells (RBCs) are one of the most susceptible biological
tissues to oxidative stress due to the presence of both high
concentration of polyunsaturated fatty acids (PUFA) in the membrane
and the oxygen transport associated with redox active hemoglobin
molecules, which are promoters of reactive oxygen species (ROS).
• Lipid peroxidation causes alteration in RBC membrane structure,
function and alteration of membrane bound receptors and enzymes.
• Phenylhydrazine (PHZ) is an antipyretic drug well known for its
ability to produce hemolysis in rats and humans by generating
superoxide anion radical and hydrogen peroxide to cause the
formation of lipid peroxidation
• Thiol compounds are the peroxidation products cause profound
alterations in the structural organization and functions of the cell
membrane including decreased membrane fluidity, increased
membrane permeability, inactivation of membrane-bound enzymes
including Na+, K+-ATPase.

54
• In almost all animal cells, including human erythrocytes, Na+, K+ -ATPase is
an essential protein transporter under normal physiological conditions.
• Decrease in erythrocyte Na+, K+ -ATPase activity associated with increased
lipid peroxidation,
• The amount of inorganic phosphate liberated is 62.21 µg of Pi liberated/min/mg
of protein as measured by ANSA reagent indicated that the membrane
architecture was preserved in-vitro.
Acetylcholinesterase assay:
• Although the physiologic functions of erythrocyte acetylcholinesterase remain
obscure, the location of this enzyme near the cell surface gives it special
significance in studies of cellular membranes and the activity alterations seen in
several haemolytic disorders may be of importance in understanding certain
basic disease process.
• Cholinesterases are enzymes, present almost exclusively in animal tissues, that
catalyze the hydrolysis of acetylcholine into acetic acid and choline
• Erythrocyte acetylcholinesterase activity alterations are seen in several
haemolytic disorders.
• PHZ intoxication leads to hemolysis resulting in severe haemolytic anaemia and
generates ROS. Our present studies, however, demonstrated that co treatment of
55
goat RBCs with PHZ and LDCP reduced ROS production.
 NO assay:
• NO is known to be a ubiquitous free-radical moiety, which is distributed
in tissues or organ systems and is supposed to have a vital role in
neuromodulation or as a neurotransmitter in the CNS.
• NO from erythrocytes comes from the fact that Hb is an avid scavenger
of NO. Notably, physiological NO concentrations range between 100
pM (or below) up to ~ 5 Nm.
• Increased intracellular concentration of NO in PHZ treated RBCs were
observed and it was caused to the accumulation of nitrite, an end
product of nitric oxide metabolism, which reacts with superoxide
radicals ultimately leading to nitro sative stress. Co-treatment of RBCs
with PHZ and fresh decoction of AD protected this increase in NO
concentration thereby indicating once again leaf decoction of Carica
papaya L. has the ability to provide protection against oxidative stress.
56
 Hexokinase Assay:
• Hexokinase is an initial enzyme of glycolysis, that catalyzes phosphorylation
of glucose by ATP to glucose-6-phosphate. Hexokinase deficiency is a
genetic autosomal recessive disease that causes Chronic Hemolytic Anemia is
caused by mutation in the Hexokinase gene. Hence, the activity of the
enzyme was determined using the LDCP against PHZ induced in goat RBCs.
• The enzyme activity was assayed by calculating the amount of glucose
phosphorylated. The lowest amount of inorganic phosphate liberated was
0.86µg of Pi liberated/min/mg of protein is reflective of extensive free radical
induced damage of PHZ. The dose dependent co-treatment of was increased
in the liberation of µg of Pi liberated/min/mg of protein by various LDCP
treated groups at the concentrations of 20,40,60,80 &100 µg/ml respectively
clearly demonsGtrated the protective effect of the substance under
investigation (LDCP) against PHZ induced Hexokinase enzyme damage.

57
 SUMMARY:
• Phenylhydrazine (PHZ) is one of the most investigated
intracellular free radical generating probes which promote
oxidative damage in erythrocytes and hence selected as model to
study the protective effect of Carica papaya L fresh leaf
decoction
• The determination of wt/mL of LDCP served to prepare
appropriate concentrations necessary for intended assays.
• The total protein assay revealed the presence of proteins which
can be molecules of interest for the inhibitory assays and can
serve as source of nutrients.
• In the current study, it was observed that the development of
oxidative stress occurred when RBCs was incubated with PHZ.

58
• The dose dependent increase in the liberation of inorganic
phosphate by various LDCP treated groups at the concentrations
of 20,40,60,80 &100 µg/ml respectively demonstrated the
protective effect LDCP against PHZ induced erythrocyte
membrane damage.
• The dose dependent increase in the liberation of thiocholine
iodide by various LDCP treated groups demonstrated the
protective effect of the substance under investigation (LDCP)
against PHZ induced erythrocyte membrane damage.
• Carica papaya having diverse anti-oxidant phytochemicals
which through unelucidated mechanism of action has significant
in-vitro protective effect on phenylhydrazine induced oxidative
damage.

59
 CONCLUSION AND FUTURE SCOPE:
• Historical accounts of certain kinds of plants and plant products reveals
that they contain pharmacologically active substances in sufficiently high
concentrations to have a drug like effect when consumed in reasonable
quantity. The in-vitro anti oxidant activity as evident by the dose
dependent increase in the liberation of inorganic phosphate in case of
hexokinase and Na+/ K+ ATPase, improved catalytic activity of
Acetylcholinesterase and decreased concentration of the free radical NO
indicates that fresh leaf decoction of Carica papaya L. has promising
anti- oxidant and anti- haemolytic activity. However these studies are
not sufficient to claim and hence rigorous, stringent battery of
pharmacological ,photochemical and bio analytical studies followed by
observational studies in humans are to be carried so as to fortify the
claimed statement of Hippocrates 400 BC ( The Father of medicine ) i.e,
“Let food be your medicine and medicine be your food”.The future work
is intended to carry out the functional status of other RBC membrane
bound as well as metabolic enzymes,.

60
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