FORMULATION & in-vitro ANTIOXIDANT ANALYSIS OF ANTI-AGEING CREAM OF CARICA PAPAYA FRUIT EXTRACT

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Indian Journal of Drugs, 2016, 4(1), 8-14 ISSN: 2348-1684
FORMULATION & in-vitro ANTIOXIDANT ANALYSIS OF ANTI-AGEING
CREAM OF CARICA PAPAYA FRUIT EXTRACT
Ritu Saini*, Aman Mittal, Vaibhav Rathi
Department of Pharmaceutics, Smt. Tarawati Institute of Bio-Medical and Allied Sciences, Roorkee, UK.
*For Correspondence:
Department of Pharmaceutics,
Smt. Tarawati Institute of Bio-
Medical and Allied Sciences,
Roorkee, UK.
ABSTRACT
Skin ageing is the outcome of constant deterioration process because of
damage of cellular DNA and proteins. It is the phase of gradual decline of
efficiency and metabolic activities. Free radical cause oxidative alterations in
collagen, elastin materials and changes in membrane characteristics and
encourage polymerization reactions. Carica papaya Linn. is one such herbal
drug commonly known as papaya belongs to the family Caricaceace. The fruit
is not just delicious and healthy, but whole plant parts, fruits, roots, bark,
peel, seeds and pulp are also known to have excellent medicinal properties
for the treatment of different diseases. Papaya is a rich source of three
powerful antioxidants vitamin C, vitamin A and vitamin E; the minerals,
manganese and potassium; the B vitamin pantothenic acid and folate and
fibers. The present study is aimed to formulate topical anti-aging cream based
on the antioxidant potential of herbal extract of papaya namely CPEAF, CPEE,
CPXE, CPHAE, CPCE and CPPEE, thus obtained were stored in desiccator and
suitable topical cream base for effective containing of fruit extract was
developed namely FOEAF, FOEE, FOXE and FOHAE. The dry extracts and
formulations were tested for in-vitro antioxidant activity. Results showed that
dried extracts CPEAF and CPXE and their corresponding formulations FOEAF
and FOXE are high in their reducing power and free radical scavenging (CPEAF
> CPXE > CPEE > CPHAE) and (CPEAF > CPXE > CPEE > CPHAE). The study could
be established that herbal creams without side effects can be used as a
barrier to protect and avoid aging of skin.
KEY WORDS: Herbal cream, Carica, Anti-ageing, Antioxidant, Free radical
Scavenging, Reducing Power, Cosmetics.
Received: 15.10.2015
Accepted: 22.03.2016
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INTRODUCTION
se of plants and plant products as medicines has been documented in the history since
centuries. Volumes of literatures have been written describing the use of various herbs,
shrubs and plants.1 Natural compounds extracted and isolated from different parts of the
plants such as leaves, fruits, stem, roots, and seeds have been shown to have admirable medicinal
value2. Skin aging is the result of continual deterioration process because of damage of cellular
DNA and protein. Aging process is classified into two different types, i.e. sequential skin aging
and photo-aging. Both types have different medical and historical features. Sequential skin aging
is widespread and probable process characterized by physiological alteration in skin function. In
the aging process keratinocytes are unable to form an efficient stratum corneum and rate of
formation from neutral lipids slows down resulting dry pale skin with wrinkle. In contrast, photo
aging is instigated by over exposure to ultra violet rays from sunlight. It is characterized by dry,
pale and thin skin, exhibiting fine wrinkles as well as deep furrows caused by the ineffectiveness
of epidermal and dermal components linked with elastosis and heliodermatitis. Medicnal plants
have already proved useful in complementary medicine.3,4 The main objective of this paper is to
evaluate the anti-ageing potential of Carica papaya fruit extract to evaluate its utility as skin
U

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rejuvenator and as an antioxidant. Ageing is the stage of gradual wearying of body efficiency and
metabolic activities after reaching a stage of maturity. Free radicals cause oxidative changes in
collagen, elastin material and membrane characteristics that induce polymerization reactions.
Carica papaya holds significant antioxidant and free radical scavenging potential. The basic
objective of this thesis is to formulate & evaluate of anti-ageing cream of Carica papaya fruit
extract in order to assess its usefulness in relieving of Oxidative stress and related conditions.
This assessment is presented from both pharmacognostic and pharmaceutical perspective. Herbal
products are very popular among people from the times immemorial due to their minimum risk
of side-effects with maximum efficacy. The data generated from the study showed that the cream
prepared from Papaya fruit extract showed significant better anti –ageing efficacy. Thus, the
cream prepared from Papaya fruit extract may prove to be an anti-ageing preparation and can be
used for retarding the symptoms of ageing. However, the results found are only directional and
further study can be made on the basis to get additional data and evidence about the Papaya fruit,
and combined effects of various botanical extracts can also be utilized on skin renewal. An
antioxidant is a molecule capable of constraining the oxidation of other molecules. Oxidation is a
chemical reaction that transfers electrons from a substance to an oxidizing agent. Oxidation
reactions can produce free radicals; these radicals could start chain reactions that cause damage
to cells. Antioxidants cut off these chain reactions by removing free radical, and inhibit oxidation
reactions. They do this by being oxidized themselves, so antioxidants are often reducing agents
such as tocopherol, ascorbic acid, thiols or polyphenols.5
Papaya, a juicy and tasty fruit, belonging to family Caricaceae is scientifically known as
Carica papaya L. It is grown in various parts of the world, including India, tropical America and
Europe. Papaya tree is basically a short lived Indian tree. Carica papaya contains many
biologically active compounds. Two important compounds are Papain and Chymopapain, which
aid in digestion. Papain is also used to treat arthritis.6
MATERIALS AND METHODS
The matured fruits of Carica papaya were collected from the local market of Roorkee city in the
month of June and were authenticated by the Botanist of department of biology, S. S. D. P. C.
Girls College, Roorkee. A voucher specimen (STIBAS/Corr/2014-15/1313) was deposited in the
Department of Botany. Cetostearyl alcohol, cetomacrogol 1000, white petroleum jelly and
propylene glycol was gift sample from Salud Care (I) Pvt. Ltd., Roorkee. All other chemicals
and solvents were of analytical reagent grade.
Preparation of Extracts:
The Successive Extraction: Firstly the outer layer and seeds of the fruits were removed and the
pulp was collected. The pulp was dried at temperature not exceeding 60ºC using hot air ovens
(Universal Hot Air Oven). About 200g of dried fruit pulp was extracted for 8 hours with
petroleum ether (40-60ºC) with soxhlet apparatus. The petroleum ether extract was filtered and
air dried. The air dried extract was repacked in the soxhlet apparatus and exhaustively extracted
with chloroform for 8 hours. The chloroform extract was filtered and again air-dried. Then
extracted plant material was repacked in the soxhlet apparatus and exhaustively extracted with
ethanol and water for 8 hours respectively. Extracts filtered and evaporated and their yield, color
and consistency were recorded.
Extraction Method for Biflavones: Powdered drug (100 g) of Carica papaya was extracted
separately in the soxhlet extraction apparatus using ethanol (95%) for 12 Hours. The resultant
alcoholic extract was then air-dried and stored in vacuum desiccators. The dried alcoholic extract

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was suspended in water. The alcoholic extract was mixed with n-hexane and the n-hexane
portion was discarded after separation. To the aqueous portion, dichloromethane was added and
the dichloromethane portion was collected and extracted with ethyl acetate. The ethyl acetate
portion was collected and solvent was completely removed. The yield of the ethyl acetate
fraction was noted. The ethyl acetate fraction was subjected to qualitative chemical test and thin
layer chromatography studies for flavonoids.7
Extraction Method for Xanthones: Powdered drug (70 g) of Carica papaya was cold
macerated (at 30-40ºC) using methanol (95%) for 24 hours. The alcoholic extract was then
mixed with n-hexane (for the removal of the fatty material may be present in the extract) and the
n-hexane portion was discarded after separation. Methanolic portion was collected and solvent
was completely removed. The yield of the ethanol was noted. The ethanolic extract was
subjected to qualitative chemical test and thin layer chromatography studies for Xanthone.8
Preparation of Topical Anti-Ageing Cream: The ingredients mentioned in the table were
weighed accurately and prepared, by melting or heating, both the phases separately to same
temperature i.e. 70ºC and mixed with continuous stirring till cream was formed. The different
extract obtained from Carica papaya fruit were added in concentration of 5% at the end.
Perfume was added after cooling of the product.9
Table 1: Formulation of Topical Anti-ageing Creams
S. No.
Name of Ingredients
QTY for 100 gm.
1
Cetostearyl Alcohol
8
2
Glyceryl Monostearate
2
3
Cetomacrogol 1000
2
4
Liquid Paraffin
5
5
White Petroleum Jelly
10
6
Propylene Glycol
20
7
Glycerine
5
8
Disodium EDTA
1
9
Ascorbic Acid
25
10
Purified Water
q. s.
11
Perfumes
q. s.
In-vitro Antioxidant activities
Reducing Power:
Reducing power is linked with antioxidant activity and serves as a significant reflection of the
antioxidant activity.10 Compounds with reducing power are electron donors and can decrease the
oxidized intermediates of lipid peroxidation processes; so that they can act as primary and secondary
antioxidants.11 The reducing power was determined by the method12of 1.0 ml sample was mixed with
2.5 ml of phosphate buffer (200 mM, pH 6.6) and 2.5 ml of potassium ferricyanide (30 mM) and
incubated at 50°C for 20 min. Thereafter, 2.5 ml of tri chloro acetic acid (600mM) was added to the
reaction mixture, centrifuged for 10 min at 3000 rpm. The upper layer of solution (2.5 ml) was mixed
with 2.5 ml of distilled water and 0.5 ml of FeCl3 (6mM) and absorbance was measured at 700 nm.
Butylated hydroxy toluene (BHT) was used as positive control.13
Determination of Hydrogen Peroxide (H2O2) Scavenging Activity:
Hydrogen Peroxide scavenging activity of sample was determined using a modification of the method
by Gow Chin Yen and Hui-Yin Chen.14 4mM solution of H2O2 was prepared in phosphate–buffered

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saline (PBS, pH 7.4). H2O2 concentration was determined using UV-VIS Spectrophotometer from
absorbance at 230nm. 20-400 μg sample corresponding to 0.10, 0.15, 0.20, 0.25ml of 1mg/ml plant
extract stock solution in 4ml distilled water were added to 0.6ml hydrogen peroxide-PBS solution.
Absorbance of H2O2 at 230nm was determined 10 minutes later against a blank solution containing
plant extract in PBS without H2O2. Different concentrations of Butylated hydroxy toluene (BHT) was
added in place of plant extract, in 4ml distilled water and the solution was added to 0.6ml H2O2
solution in PBS. Absorbance was determined 10 minutes later against a blank solution similar to that
above.15 Percentage free radical scavenging activity was calculated using the formula:
% SA = 𝐴𝐶−𝐴𝐸
𝐴𝐶
𝑋100
Where AC=Absorbance of control, AE=Absorbance of Extract, % SA=Percentage scavenging activity
Free radical scavenging assay:
Effect of the sample extracts on DPPH radical was measured using this method. Amount of 200 µl of
the sample extract (0.62 – 4.96 mg/ml) or ascorbic acid (0.04 – 1.28 mg/ml) were added to 1 ml of 0.1
mM DPPH in 80% methanol. The mixture was shaken vigorously and left to stand in dark room for 30
min at room temperature. Absorbance of the solution was measured using UV-VIS Spectrophotometer
at 517 nm with deionized water as blank.13, 16 the capability of sample to scavenge the DPPH radical
was calculated according to the equation as follows:
% SA = 𝐴𝐶−𝐴𝐸
𝐴𝐶
𝑋100
Where AC=Absorbance of control, AE =Absorbance of Extract, % SA=Percentage scavenging activity
RESULT AND DISCUSSION
Reducing Power
The results of this study shows that the reducing power of the FOHAE was less active than all extracts
and FOEAF, as defined by the intensity of UV absorption even its highest concentration was less
active than the lowest concentration (25µg/ml) of BHT. (Figure 1, Table 2), the ethyl acetate fraction
shows intense absorptions as compared to other extracts. FOEE showed significant absorption only at
higher concentration (400µg/ml), FOXE shows higher activity than FOEE and FOHAE but lesser than
the BHT and ethyl acetate fraction. In an overall reducing power analysis the tests can be arranged as.
BHT > FOEAF > FOXE >FOEE >FOHAE
Determination of Hydrogen Peroxide (H2O2) Scavenging Activity:
The results of this study shows that the reducing power of the FOHAE was less active than all extracts
and FOEAF, as defined by the intensity of UV absorption even its highest concentration was less
active than the lowest concentration (25µg/ml) of BHT. (Figure 2, Table 3), the ethyl acetate fraction
shows intense absorptions as compared to other extracts. FOEE showed significant absorption only at
higher concentration (400µg/ml), FOXE shows higher activity than FOEE and FOHAE but lesser than
the BHT and ethyl acetate fraction. In an overall reducing power analysis the tests can be arranged as.
BHT > FOEAF > FOXE >FOEE >FOHAE
Free radical scavenging assay
Effect of the standard (Ascorbic acid) and sample extracts on DPPH radical is shown in Table 4 and
corroborated by Figure 3 at concentrations as low as 0.62 mg/ml, where STD had 34.33 % efficiency,
FOEAF mopped up to 28.33 % as compared to 19.67 %, 15.22 % and 7.49 % for FOEE, FOXE and
FOHAE respectively. Similarly, STD, FOEAF, FOEE, FOXE and FOHAE scavenged 78.89 %, 72.31
%, 53.34 %, 45.36 % and 27.42 % respectively at a maximum concentration of 4.96 mg/ml.
Conc.
(µg/ml)
BHT
FOEAF
FOXE
FOEE
FOHAE

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400
1.62±0.01
1.33±0.015
0.66±0.02
0.96±0.04
0.18±0.015
200
0.72±0.011
0.62±0.02
0.53±0.026
0.54±0.015
0.11±0.015
100
0.69±0.015
0.29±0.02
0.24±0.015
0.35±0.57
0.08±0.011
50
0.51±0.13
0.24±0.015
0.19±0.02
0.21±0.01
0.02±0.005
n=3±SD
Table 2: Reducing Power of BHT (Standard) and different formulation of Topical Anti-ageing
Cream
Conc.
(mg/ml.)
Scavenging activity (in percentage)
BHT
FOEAF
FOEE
FOXE
FOHAE
0.0250
41.73±0.20
38.72±0.15
31.48±0.10
29.57±0.12
10.23±0.14
0.0375
64.73±0.15
58.14±0.17
50.14±0.17
47.29±0.12
23.67±0.12
0.0500
82.35±0.15
73.49±0.19
66.84±0.15
67.03±0.10
31.82±0.18
0.0625
92.40±0.20
79.83±0.10
75.44±0.17
69.55±0.10
39.58±0.15
n=3±SD
Table 3: H2O2 Percentage scavenging of BHT (Standard) and various formulation of Topical
Anti-ageing Cream
Conc.
(mg/ml.)
Scavenging activity (in percentage)
STD
FOEAF
FOXE
FOEE
FOHAE
0.62
34.33±0.17
28.13±0.17
19.67±00.15
15.22±0.20
7.49±0.05
1.24
43.26±0.15
34.69±0.10
30.23±0.15
23.668±0.21
11.56±0.02
2.48
66.31±0.10
61.11±0.20
46.21±0.20
32.16±0.15
18.59±0.10
4.96
78.89±0.18
72.31±0.20
53.34±0.10
45.36±0.17
27.42±0.11
n=3±SD
Table 4: Free radical scavenging of Standard (Ascorbic acid) and various formulation of Topical
Anti-ageing Cream
Figure 1: The graph between Concentration (µg/ml) & Absorbance at 700 nm
0
0.5
1
1.5
2
0100 200 300 400 500
Absorbance at 700 nm
Concentration in µg/m
The Graph between Concentration (µg/ml) & Absorbance
(700 nm)
BHT
CPEAF
CPEE
CPXE
CPHAE

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Figure 2: The Graph between Concentration (mg/ml) & % Scavenging
Figure 3: The Graph between Concentration (mg/ml) & % Scavenging
CONCLUSION
From all results and considerations it can be determined that the cream prepared from Carica papaya
fruit extract showed significantly better anti-ageing efficacy as compared to the control. The cream
prepared from Carica papaya fruit extract is an ideal oil-in-water cream with good consistency,
smooth & shining texture, good stability and may prove to be an anti-ageing preparation and can be
used for retarding the symptoms of ageing and showing skin-renewal activity. The outcomes obtained
in this study are only directional and further investigations can be made on this ground, to get
additional data and information about Carica papaya fruit, and combined effects of various botanical
extracts can also be studied on skin renewal.
ACKNOWLEDGEMENT
Authors are thankful to Smt. Tarawati Institute of Pharmacy, Roorkee for continuous moral support
and providing library facility and also thankful to Salud Care (I) Pvt. Ltd., Roorkee, India
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