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Sun Screen Efficacy of Punica granatum (Pomegranate) and Citrullus colocynthis

(Indrayani) Seed Oils

Article · October 2016

DOI: 10.22192/ijarbs.2016.03.10.027

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 Int. J. Adv. Res. Biol. Sci. (2016). 3(10): 198-206

International Journal of Advanced Research in Biological Sciences

ISSN: 2348-8069
www.ijarbs.com
DOI: 10.22192/ijarbs Coden: IJARQG(USA) Volume 3, Issue 10 - 2016
Research Article

DOI: http://dx.doi.org/10.22192/ijarbs.2016.03.10.027

Sun Screen Efficacy of Punica granatum (Pomegranate) and

Citrullus colocynthis (Indrayani) Seed Oils

Janaky Ranjithkumar, Akhila Sameesh, Hari Ramakrishnan, K.

Kaleesuwari Refinery Private Limited, Department of Research and development,
No. 5, Mambakkam Road, Vengaivasal, Chennai, Tamil Nadu, INDIA, 600 126
Author for Correspondence: Hari Ramakrishnan. K, E-mail: [email protected]

Abstract
With increasing awareness about skin cancer and its relation to UV radiation, there is much focus on sunscreen agents and
products. Sunscreens provide protection by absorbing, reflecting and / or scattering the radiation. Sunscreens are rated according
to their effectiveness by sun protection factor (SPF). The present study evaluates ultraviolet (UV) absorption ability of Citrullus
colocynthis (Bitter Apple, Indrayani) seed oil, Punica granatum (Pomegranate) seed oil and few other vegetable fixed oils. The
in-vitro SPF is determined according to the spectrophotometric method of Mansur et al. The UV absorbances of alcoholic
solutions of the oils were studied by UV spectrophotometer in the range of 290 nm to 320 nm at 5 nm increments. The in-vitro
test results show that Pomegranate seed oil shows an SPF at par with synthetic sunscreens at 1.0 % level, with Indrayani seed oil
following closely.

Keywords: Sun protection Factor (SPF), Indrayani (Citrullus colocynthis) seed oil and Pomegranate (Punica granatum)
seed oil.

Introduction
Sun is the source of energy. It is vital for life. but not all, of the UV B rays. With depleting ozone
Exposure to UV light has number of different effects layer, protection from UV B rays is gaining great
on the human body. UV radiation is absorbed by importance (Shantanu Kale et al., 2010).
photoreceptors in the skin, after which substances that
suppress cell-mediated immunity may be released. As The UV A and UV B radiations reaching earth’s crust
a consequence, both local and systemic immune are of lower energy. UV A radiation’s photo-
functions may be modulated. Recent evidence biological effects are cumulative (long term) effects.
suggests a beneficial effect of UV light on the severity UV A radiation penetrates deep into the dermis and
of some autoimmune diseases, such as multiple beyond, i.e. 20 % to 30 % reaches the dermis. UV A
sclerosis (David S, Strayer, 2015). According to its has been shown to cause cell damage (Wondrak et al.,
physiological activity, UV radiation is subdivided into 2003, Scharffetter et al., 1997), connective tissue
UV A (400 nm to 320 nm), UV B (320 nm to 280 nm) damage (Lorraine et al., 1985) and induce premature
and UV-C (280 nm to 100 nm) (De Polo,1998). UV ageing. UV B radiation is energy rich and produces
C, the high energetic electromagnetic radiation, lethal intense short range and long range patho-physiological
to all living organism, is absorbed by the ozone layer photo-damage to skin. About 70 % is reflected by the
located in the stratosphere. It also blocks most, horny layer, 20 % penetrates into deeper layers of the
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 Int. J. Adv. Res. Biol. Sci. (2016). 3(10): 198-206
epidermis and 10 % reaches the dermis (De Polo, reflect UV energy back into the environment. Only
1998). The short terms effects of UVB radiation are fifteen chemical sunscreens are approved by US FDA
immediate tanning or immediate pigment darkening for use in Sunscreen products, with use level
(IPD) (Rainer et al., 2009) and solar erythema (sun restrictions (Steven Q Wang and Henry W, 2011).
burn). The sun burning effective energy curve has a
maximum at about 305 nm. UV B sunscreen agents Since no single agent effectively provides adequate
must possess an absorption maximum at or near this protection from both UVA and UVB radiation, nearly
wave length (Documentation of Solar Light, Sayre all commercially available sunscreen products contain
R.M, 1992). Exposure to UV radiation on the skin agents from both groups. Two or more sunscreen
results in mutagenic effects (Ouhtit et al., 1998). It active ingredients may be combined with each other in
can damage DNA and is a leading cause of skin cancer a single product when used in the concentrations
(David S, Strayer, 2015). approved by the US FDA for each agent. Each
individual active ingredient must contribute a
Photo-protection is therefore focused on protecting the minimum SPF of at least 2 to the finished product.
skin from the damage that occurs due to UV exposure. The finished product should have a maximum SPF of
Increasing awareness of the damaging effects of not less than the number of sunscreen active
sunlight has led to increased need for adequate photo- ingredients used in the combination multiplied by two
protection. Primary prevention includes a regimen (Nathalie and Darell, 2006). Exposure to UV radiation
consisting of effective sunscreen and protective stimulates endogenous production of vitamin D in the
clothing. Clothing is the best way to get protection skin. UV wavelengths between 270 nm and 300 nm,
from these UV A and UV B rays. However, the UV B region, result in production of a precursor
depending on circumstances, sunscreen products need for vitamin D from a cholesterol derivative (David S,
to be used. Sunscreens work primarily through two Strayer, 2015).
mechanisms: (i) absorption and (ii) scattering and
reflection of UV energy. Many current sunscreen The efficacy of a sunscreen agent or product is
products contain sunscreen agents that work through expressed by Sun Protection Factor (SPF) for UV B.
both mechanisms in terms of UV protection. SPF is defined as the UV energy required to produce a
Chemical Sunscreens and Physical Sunscreens are the Minimal Erythemal Dose (MED) in protected skin
two types of sunscreens used in Sunscreen products divided by the UV energy required to produce the
(Brummitte et al., 2012). Physical sunscreens, the same MED in unprotected skin (Nadim A, 2005).
barrier substances, are particles that mainly scatter and

Minimal erythema dose in sunscreen-protected skin

SPF =
Minimal erythema dose in non-sunscreen-protected skin
Mansur et al., 1986 developed a very simple
MED is defined as the lowest time interval or dosage mathematical equation which substitutes the in- vitro
of UV light irradiation sufficient to produce minimal method proposed by Sayre et al., 1979 utilizing UV
perceptible erythema (redness of skin) on unprotected spectrometry and the following equation:
skin. The higher the SPF, the more effective is the
product in preventing sun burn.

There are various methods available for the

determination of SPF. It can be determined by in-vivo Where
or in-vitro and it is ideally determined by photo-testing
on human volunteers. Both the in-vitro and in-vivo EE – Erythemal Effect spectrum
methods are approved by US FDA and COLIPA I – Solar Intensity spectrum
(Allen MW , 2007). Though photo-testing on humans, Abs – Absorbance of sunscreen product
an in-vivo method, is useful and precise, it is time CF – Correction Factor
consuming, complex and expensive. So, effort has The value of EE x I are constants and were determined
been devoted to the development of in-vitro by Sayre et al and the normalized values are presented
techniques. in Table 1 (Sayre et al., 1979 )

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 Int. J. Adv. Res. Biol. Sci. (2016). 3(10): 198-206
Table 1 – Normalized product function used in calculation of SPF

Wavelength EE x I
 nm (normalized)
290 0.0150
295 0.0817
300 0.2874
305 0.3274
310 0.1864
315 0.0839
320 0.0180
TOTAL 1
EE – Erythemal Effect Spectrum; I – Solar Intensity Spectrum
Mansur equation has been used for the determination radiation and also as predicted by Woodward–Fieser
of SPF of various sunscreen agents, synthetic rule (Wagemaker et al., 2011).
molecules, natural extracts, oils, emulsions, etc. For
example, SPF value of Usnea rocellina (Jose et al., CH2CH-CHCH2 CH2CH-CHCH-CH2-CH3
2015) Musa accuminata, Psidium guaja & Pyrus 1,3-Butadiene 1,3-Hexadiene
communis (S. Imam and Mahmood , 2015) Zingiber max 290 nm max 350 nm
officinale (Manoj ,2014) , Murraya koenigii (Rekha et
al., 2010) Psidium guaja (Vandana et al., 2016) As per Woodward–Fieser rule, and based on Structure
Portulaca oleracia (Vandana et al., 2015) Opuntia Activity Relationship, conjugated fatty acids present in
ficus-indica (Cinthya et al, 2014) Corn cob (Zea mays the triglyceride form also should exhibit absorbance in
et al., 2013), Garcinia mangostana (Liandhajani et al., the UV range (Wagemaker et al., 2011). If they
2013), Calendula officinalis (Mishra et al., 2012) possess UV radiation capabilities, they will be of use
were evaluated using Mansur equation. in sunscreen preparations.
Jyostna A S Suryavanshi , 2016 published the SPF
value of few herbal oils using Mansur equation. We identified Indrayani (Bitter Apple, Citrullus
However, the concentration of the herbal oils tested colocynthis) seed oil and Pomegranate (Punica
was not mentioned. Chanchal Deep Kaur and granatum) seed oil as having potential for high SPF
Swarnalata Saraf (Chanchal and Swarnlata, 2010) value, based on their fatty acid profile.
determined the SPF of Olive, Coconut, Castor,
Almond, Mustard, Sesame and a list of essential oils in Citrullus colocynthus (of the family Cucurbitaceae),
aqueous ethanolic solutions. In this, due to the also known as bitter apple, Indrayani, is a fruit-bearing
presence of water, the aqueous alcoholic solution of plant. It is traditionally known both as a medicinal
the fixed oils clarity could not be obtained. This and toxic plant (Jouad , 2001). In Folklore, it is used
restricts the free evaluation of various concentrations. for the treatment of rheumatism, stimulating the
They have not tested in pure ethanolic solution. immune system, tuberculosis, diabetes, and analgesic
(Daoudi , 2013). Oral ingestion of the fruit prescribed
The UV absorbance of compounds with conjugated for diabetes (Huseini , 2009). Petroleum ether extract
and extended conjugated double bonds could be of the fruits of Citrullus colocynthis shown to induce
predicted by Woodward Fieser rule. The wavelength hair follicle density (Dhanotia , 2011).
will have bathochromic or hypsochromic shift,
depending on the extension of conjugation, presence Punica granatum L., commonly known as
of electron donating and withdrawing groups. Pomegranate, belongs to Punicaceae family, is one of
Woodward–Fieser rule is empirically derived, which the most ancient edible fruits, widely grown in India
helps to predict the absorption maxima (λmax) in an and Mediterranean regions. Traditionally it has been
ultraviolet–visible spectrum of a given compound esteemed as a food and medicine in India (Nadkarni,
(Valdas and Gerald, 2015). 2007). Pomegranate, known for its high antioxidant
potential, has been used for medicinal properties for
On the basis of Structure Activity Relationship, dienes centuries (Syed et al., 2007).
and conjugated dienes should absorb UV radiation.
1,3 - Butadiene and 1,3 – Hexadiene are absorbing UV
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 Int. J. Adv. Res. Biol. Sci. (2016). 3(10): 198-206
Pomegranate seed oil (PSO) represents between 12 to rheumatoid arthritis and coronary heart disease
20 % of the seed. The seed oil is a triglyceride. The (Boussetta et al., 2009).
predominant fatty acid is Punicic Acid (Mukherjee and
Bhattacharyya , 2002) a conjugated fatty acid with These two oils are fixed oils, meaning triglycerides,
three double bonds. PSO has been reported to exhibit along with unsaponifiables in minor quantity, like any
in-vivo antioxidant and anti-inflammatory activities, other vegetable derived fixed oil.

Figure 1: The structure of Punicic acid is given below along with Linolenic and Linoleic acids

Punicic Acid Linolenic Acid Linoleic Acid

Triglycerides are glycerol molecule esterified with Pomegranate (Punica granatum) Seed Oil was
three molecules of fatty acids. The fatty acids present obtained from Cyrus Enterprises, (Mugappair East,
in the triglyceride vary from oil to oil. They may be Chennai, Tamil Nadu, India).
saturated, monounsaturated or polyunsaturated, with
their chain length normally varying from C4 to C22, All the other vegetable oils were bought from the
unsaturation usually vary from 1 to 6. In the market:
polyunsaturated molecules, the double bonds may be
conjugated or unconjugated. The conjugation is (1) Sesame Oil (Kaleesuwari Refinery)
responsible for the UV absorption property. This (2) Coconut Oil (Eldia Pure™)
conjugation allows the molecule to absorb high-energy (3) Sunflower Oil (Gold Winner™)
ultraviolet rays and release the energy as lower-energy (4) Extra Virgin Olive Oil (Cardia™)
rays (Tentative Final Monograph, 2009). (5) Refined Olive Oil (Kaleesuwari Refinery)
(6) Groundnut Oil (Gold Winner™)
Punica granatum seed oil and Citrullus colocynthis (7) Canola Oil (Borges™)
seed oil, along with few more vegetable oils, were (8) Corn Oil (Kaleesuwari Refinery)
screened for UV absorbance. For comparison, three (9) Avocado Oil (free from Textron)
US FDA approved synthetic sunscreens (10) Sweet Almond Oil (Dabur Badam Ki Tail)
(Benzophenone 3, Octocrylene and Ethylhexyl
Salicylate) were also included in the study. Neo Heliopan BB (Benzophenone 3), Neo Heliopan
303 (Octocrylene) and Neo Heliopan OS
Materials and Methods (Ethylhexyl Salicylate) were received free from M/s
Symrise.
Reagents: Ethanol (Merck) Analytical Grade
Extraction of Oil: The dried seeds were powdered,
Apparatus: UV Double Beam Spectrophotometer, filled in thimbles and extracted with Diethyl ether as a
equipped with quartz cell. solvent using soxhlet. The filtered extracts were dried
over anhydrous sodium sulphate. The dried extracts
Sample Preparation / Sourcing: were distilled off using thin film evaporator. The final
traces of solvents were removed using dry nitrogen.
Sourcing: They were kept at -15 C in hermetically sealed amber
coloured culture tubes.
Indrayani (Citrullus colocynthis) seed were obtained
from ASP Herbals, (Parrys, Chennai, Tamil Nadu,
India).
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 Int. J. Adv. Res. Biol. Sci. (2016). 3(10): 198-206

UV Absorbance: Results and Discussion

1.0 g of all samples was weighed, transferred to a 100 All the oils were tested for their physiochemical
ml volumetric flask, made up to 100 ml using ethanol, properties and fatty acid profile. The fatty acid profile
followed by mixing in sonicator for 8 minutes. The was determined by converting the triglycerides to their
clear solution was further diluted to 0.1 %, 0.5 % and corresponding FAME (fatty acid methyl ester) and
0.05 % solutions in volumetric flasks. The aliquots was analyzed by Gas Chromatography. The data is
absorbance was measured between 290 nm and 320 presented in Table 2. The in vitro test results (Table
nm at 5 nm increments. The obtained values were :3) show that Pomegranate Seed Oil shows an SPF
multiplied with the respective EE (λ) values. All equivalent to Synthetic Sunscreens at 1.0 % level, with
experimental absorbance values were collected in Indrayani Seed Oil following closely. Their results
triplicate and the average values were taken for SPF were good even at 0.50 % level. Corn, Canola, Extra
calculation. Virgin Olive, Refined Olive, Ground Nut and Sesame
oils, while having respectable SPF at 1.0 % level, they
fail at 0.50 % level (Figure 2).

Figure 2: Bar Graph of SPF of vegetable oils

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 Int. J. Adv. Res. Biol. Sci. (2016). 3(10): 198-206

Table 2 - Physicochemical and Fatty acid profile of vegetable oil

Physiochemical Properties
PSO CCSO RBO Coconut Sunflower EVOO Groundnut Canola Corn ROO SSO
Colour (Lovibond,
12 31 15 2 8 35 8.5 2.5 5.0 3 11.5
Y+5R)
Acid Value (mg KOH /
3.867 6.36 0.2 3.53 0.2 0.46 0.08 4.22 0.1 0.2 6.3
g)
Saponification Value
193.23 201.23 192 251.69 190.8 190.16 192.16 190.75 191.08 190 190.83
(mg KOH / g)
Free Fatty Acid 3.60 4.75 0.10 1.26 0.10 0.23 0.04 0.03 0.05 0.1 3.15
Iodine Value (g Iodine /
160 118.64 97 8.59 128 81.98 92.13 87.2 114.65 83 107
100 g)
Peroxide Value (meq
3.60 13.3 1.00 1.26 0.65 6.26 2.14 4.50 0.89 1.25 16.2
oxygen / Kg)
Specific Gravity at 20 0.9168(3 0.915 0.9115 0.918 (20 0.916(20 0.912(20
0.940 0.912 (20 C) 0.910(20 C) 0.910 0.915 (20 C)
C 0 C) (20 C) (30 C) C) C) C)
Refractive Index at 20 1.4650(4 1.4608 1.4610 1.4670 (40 1.4621(40 1.4652(40 1.4679(40 1.4656(40
1.5160 1.4606 1.4626
C 0 C) (40 C) (40 C) C) C) C) C) C)
Moisture negligible 0.01 0.01 0.02 0.01 0.04 0.01 0.001 0.12 0.01 0.06
Fatty Acid profile by Gas Chromatograph
Caproic Acid (C6:0) NIL NIL NIL 0.694 NIL NIL NIL NIL NIL NIL NIL
Caprylic Acid (C8:0) NIL NIL NIL 7.986 NIL NIL NIL NIL NIL NIL NIL
Capric Acid (C10:0) NIL NIL NIL 5.424 NIL NIL NIL NIL NIL NIL NIL
Lauric Acid (C12:0) NIL NIL NIL 46.519 NIL NIL NIL NIL NIL NIL NIL
Myristic Acid (C14:0) 0.010 0.319 19.752 0.048 0.011 0.056 0.013 0.040 0.009 0.015
Palmitic Acid (C16:0) 2.708 11.723 19.991 8.417 5.730 10.724 13.326 4.421 13.574 10.150 9.353
Stearic Acid (C18:0) 2.282 7.823 1.941 2.728 3.703 3.994 4.149 1.52 2.135 3.587 6.511
Oleic Acid (C18:1) 5.739 20.538 41.363 6.607 27.873 76.660 41.033 60.97 0.207 78.463 43.198
Linoleic Acid (C18:2) 5.808 58.884 30.834 1.680 60.890 5.924 32.784 19.557 47.334 5.153 39.359
Linolenic Acid (C18:3) 0.009 0.080 0.666 0.12 0.033 0.628 0.097 9.532 0.807 0.420 0.258
Arachidic Acid (C20:0) 0.433 0.380 0.843 0.077 0.308 0.425 4.802 0.592 0.622 0.506 0.700
Gadoleic Acid (C20:1) 0.486 NIL 0.521 0.042 0.152 0.237 0.909 1.442 0.355 0.235 0.184
Punicic Acid (C18:3,
81.879 NIL NIL NIL NIL NIL NIL NIL NIL NIL NIL
conjugated)
Eicosenoic acid (C20:1) NIL NIL NIL NIL NIL NIL NIL NIL NIL NIL
Behenic Acid (C22:0) NIL 0.136 0.284 0.015 0.817 0.111 3.949 0.347 0.178 0.113 0.152
Erucic Acid (C22:1) NIL NIL NIL NIL NIL NIL 0.084 0.715 NIL NIL NIL
Lignoceric Acid (C24:0) 0.066 0.138 0.496 0.034 0.300 0.051 1.471 0.11 0.245 0.049 0.108

PSO – Cold Pressed Pomegranate Seed Oil, CCSO – Bitter Apple Seed Oil, RBO – Refined Rice Bran Oil (Orysa), CNO - Coconut Oi (Eldia Pure), SFO –Sunflower
Oil (Gold Winner), EVOO – Extra Virgin Olive Oil (Cardia), GNO – Groundnut Oil (Gold Winner), Canola – Canola Oil (Borges), Corn – Corn Oil, ROO – Refined
Olive Oil (Cardia), SSO – Sesame Oil

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 Int. J. Adv. Res. Biol. Sci. (2016). 3(10): 198-206

Table 3 - SPF of Vegetable Oils determined using Mansoor’s equation

SPF as determined using Mansoor Equation
Ethanolic solution (in 1.00 0.50 0.10 0.05
%) % % % %
Benzophenone 3 21.28 21.27 21.23 20.23
Octocrylene 21.00 20.98 20.94 20.87
Ethylhexyl Salicylate 20.79 20.79 20.70 20.48
Indrayani Seed Oil 17.37 12.43 3.49 2.28
Pomegranate Seed Oil 21.16 17.08 10.54 7.93
Canola Oil 13.02 1.51 1.19 1.08
Extra Virgin Olive Oil 11.71 0.99 0.90 0.91
Refined Olive Oil 10.21 1.23 0.97 0.76
Corn Oil 15.22 2.61 0.77 0.53
Sesame Oil 15.12 3.65 1.19 0.95
Groundnut Oil 13.30 0.93 0.48 0.39
Sunflower Oil 9.82 1.31 0.52 0.28
Coconut Oil 1.36 0.60 0.62 0.48
Sweet Almond Oil 3.70 0.14 0.08 0.17
Avocado Oil 0.08 0.07 0.04 0.03

Following the numbering from the methyl end, the %, Pomegranate seed oil shows an in vitro SPF of 8
double bonds are at carbon atoms 5, 7 and 9 positions and Indrayani seed oil SPF of 2.3, they shall be
(Figure 1). As per configuration, Punicic Acid has 5- evaluated in sunscreen preparations, intended for skin
cis, 7-trans and 9-cis conformation. It has a 1,3,5- and hair care. In addition to their sun protection
hexatriene backbone with two alkyl groups at the two properties, being triglycerides, they also act as good
ends (i.e. at C5 and C10). As per Woodward Fisher emollients / spreading agents for other sunscreens.
rule, UV absorbance maxima of 247 nm (base wave
length) + (2 x 5nm) [for the two alkyl groups] = 257 Conflict of Interest: The authors declare that they
nm could be predicted for this molecule. have no conflict of interest.
The result of Pomegranate seed oil obtained is as
expected and also as calculated by Woodward Fieser
Acknowledgement: The authors are thankful to
the Directors - Mr. Arun, M., and Mr. Ashok, K., and
rule. With respect to Indrayani seed oil, the results
to Chief Operating Officer Mr. Anurag Lodha of
were more than expected. Indrayani Seed contains
Kaleesuwari Refinery Pvt. Ltd. for providing constant
neither any conjugated fatty acids nor any molecules
inspiration for this work.
in the unsaponifiable part bearing chromophoric group
possessing UV absorbance properties. More research
will be continued with Indrayani seed oil to find out References
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DOI:10.22192/ijarbs.2016.03.10.027

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Janaky Ranjithkumar, Akhila Sameesh, Hari Ramakrishnan, K. (2016). Sun Screen Efficacy of Punica
granatum (Pomegranate) and Citrullus colocynthis (Indrayani) Seed Oils. Int. J. Adv. Res. Biol. Sci. 3(10):
198-206.
DOI: http://dx.doi.org/10.22192/ijarbs.2016.03.10.027

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