FIELD OF THE INVENTION
This invention relates to the Preparation, Evaluation and Characterization of a formulation for its
Photoprotective and Radio decontamination activity. The present work aims at formulating a
suitable topicalsemi-solid formulation of rosmarinic acid for effective photo-protection as well as
radio-decontamination purposes.
FIELD OF THE INVENTION
In humans, skin is the largest organ of the integumentary system covering the entire body. It is
made up of multiple layers of ectodermal tissue. It guards the underlying muscles, bones,
ligaments and internal organs in addition to serving as a protective shield against heat, light,
injury, infection etc. Skin also regulates body temperature, stores water and fat and prevents loss
of water and entry of bacteria. However, the integrity of skin is adversely affected on exposure to
ultraviolet radiations.
The major acute effects of UV irradiation on normal human skin comprise sunburn inflammation
(erythema), tanning, and local or systemic immunosuppression. At the molecular level, exposure
to UV irradiation causes DNA damage such as cyclobutane pyrimidine dimers and (6-4)
photoproducts formation. Chronic exposure to UV irradiation leads to photoaging,
immunosuppression, and ultimately photo-carcinogenesis. Photo-carcinogenesis involves the
accumulation of genetic changes as well as immune system modulation and ultimately leads to
the development of skin cancers.
UV radiations can be effectively prevented by using photo-protective agents suitably
incorporated in dermatological preparations. Synthetic products such as benzyl salicylates and
salicylates derivatives, benzyl cinnamates and cinnamates derivatives, p- aminobenzoic acid,
zinc oxide and titanium dioxide etcare potential UV rays protecting agents. However, topically
applied sunscreen blocks UV rays as long as it does not penetrate into the skin. If the sunscreen
is absorbed into the skin, it prevents sunburn but increases the amount of free radicals, which in
turn increases risk for malignant melanoma. The harmful effect of photo-excited sunscreen in
living tissue has been shown in many photo-biological studies(Armeni et al, 2004; Knowlandet
al, 1993; Mosley et al, 2007). So a good alternative to all such problem is use of herbal agents.
3
Not only does UV cause significant changes in the body, radiations from radionuclides too may
cause serious health consequences. Such radiations include beta, gamma and alpha rays. The
main effect of such radiation is ionisation of the atoms in the absorbing medium. Thus, when
cells are irradiated, ionisation of one or more of the atoms of DNA molecules will occur. Effects
include breakage of the chains of molecules comprising DNA and links between chains.
In many cases, the cell is able to repair the damage, but not always. When the damage cannot be
repaired, the affected cell is left with altered or damaged genetic information, compared with the
unaffected cells. All descendants of that cell will contain altered or damaged information.
The direct attack of radiation on the structure of DNA is not the only means by which radiation
can affect cells. Majority of the human body (about 60%) is made up of water. Ionizing effects
of radiation on water can lead to an indirect attack on DNA via generation of hydrogen oxide
molecule. Hydrogen oxide molecule can react with DNA and damage the cells alongwith genetic
information contained therein. Therefore, cells can be subject to an indirect attack due to the
action of radiation on body water, as well as from the direct effects of ionisation at the site of the
DNA.
Radionuclides are extensively used these days for power generation, in field of medicine,
industry, agriculture and health. The phenomenal growth in application of radio isotope has
helped in approving the quality of life of human race. It is a matter of fact that application of
nuclear energy/radio isotope are among the world’s best regulated ones, with the highest safety
record because of the best safety practices and standards followed in these applications
throughout the world. Inspiteof all such care, the incident of radio contamination cannot be ruled
out. Radio contamination implies unwanted toxic radiological material in/on the body of affected
individuals. At such a time of nuclear and radiological emergency, the first step is to remove the
victim from the contaminated site followed by removal of toxic radiological material from the
body of contaminated victim.
The most important property of a compound for radio-decontamination is metal ion chelation
activity. This is made possible if the compound possess inherent capability to bind with metal
ion. The commonly used agents for decontamination purposes are DiethylenetriaminePenta4
acetic acid (DTPA), Ethylenediaminetetraacetic acid (EDTA), Nitrilotriacetic acid (NTA) etc.
These agents functions by forming a complex with the metal and helps in its elimination from the
body surface.Agents which can be used for radio-decontamination purposes must possess good
chelating efficacy for radionuclides. Also, they must be non-toxic to the body, must be easily
available and rapid in action. The above listed compound however suffers from problem with
regard to their cost and skin irritation on prolonged usage. Use of herbal compounds can be
effectively made because the herbal nature of these compounds has added advantage of being
safe on prolonged usage.
SUMMARY OF THE INVENTION
India has been known for its rich herbal heritage since ages. The activity of these plants is
diverse. These plants are source of various therapeutically active substances having activity such
as immuno-modulatory, anti-inflammatory, antibacterial, antifungal and many more. Nature has
given us a plethora of compounds of immense potential such as anti-oxidants, photo-protector
and radio protector. Polyphenols are one of such compounds which have all these activities.
Some of them are rosmarinic acid, carnosic acid, carnosol etc. Rosmarinic acid, one of the
polyphenol has immense potential for protection against UV rays owing to its free radical
scavenging activity and inducing body’s own defense mechanism by regulating tyrosinase
activity and stimulating melanin production. Thus, it can act as both exogenous as well as
endogenous photo-protector. It has been further found that RA is having γ rays protectant
activity. Taking into consideration the structure of rosmarinic acid consisting of multiple –
COOH and –OH group co-existing, this can act as site for binding with metal ions. Thus,it can be
a potential candidate for radio-decontamination purposes as well (Table 1).
Different properties of rosmarinic acid relevant to photo-protection and radio-protection:
Sr.
No
Properties Description References
1 UV A protective
activity
UVA exposed human
keratinocytes cell line (HaCaT)
were treated with rosmarinic
PsotovaJ, SvobodovaA, Kolarova
H, Walterova D. 2006.
Photoprotective properties of
5
acid. RA showed reduction in
UVA caused decrease in cell
viability monitored by neutral
red retention and by LDH
release into medium. Also it
suppressed UV-A induced
ROS production and reduced
DNA damage.
Prunella vulgaris and
rosmarinicacid on human
keratinocytes.JPhotochemPhotobiol
B. 84(3):167-74
2 UV B protective
activity
Pre and post-treatment of
HaCaT cells with Rosmarinic
acid reduced DNA breakage
together with the apoptotic
process on exposure to UV B
rays. Rosmarinic acid also
significantly eliminated ROS
production and diminished IL-
6 release and reduced LDH
release into the medium.
Vostalova J, Zdarilova A,
Svobodova A. 2010. Prunella
vulgaris extract and rosmarinic acid
prevent UVB-induced DNA
damage and oxidative stress in
HaCaT keratinocytes. Arch
Dermatol Res. 302: 171–181
2 Anti-oxidant
activity
By using TBARS method and
checking malonyldialdehyde
production due to linoleic acid
peroxidation.Trolox equivalent
antioxidant capacity (TEAC)
method is also used.
Bano MJD, Lorente J, Castillo J,
Benavente-Garcia O, Rio JAD,
Ortuno A, Quirin KW, Gerard D.
2003. Phenolic Diterpenes,
Flavones, and Rosmarinic Acid
Distribution during the
Development of Leaves, Flowers,
Stems, and Roots of Rosmarinus
officinalis. Antioxidant Activity. J.
Agric. Food Chem. 51: 4247-4253.
Campillo, M.S, Gabaldon J.A.
2008. Rosmarinic acid, a photoprotective
agent against UV and
6
other ionizing radiations. Food
Chem. Toxicol. 47: 386-392.
3 Radio-protective
activity
Evaluating the reduction in the
frequency of micronuclei (MN)
incytokinesis-blocked cells of
human lymphocytes before and
after γ-ray irradiation.
Bano MJD, Castillo J, Benavente-
Garciä O, Lorente J, Martin-Gil R,
Acevedo C, Alcaraz M. 2006.
Radioprotective-Antimutagenic
Effects of Rosemary Phenolics
against Chromosomal Damage
Induced in Human Lymphocytes
by γ-rays J. Agric. Food Chem. 54:
2064-2068
4 Photo-protective Rosmarinic acid acts as
exogenousand endogenous
photo-protector, in the first
case acting asfree radical
scavenger and in the second as
an inducer of the body’sown
endogenous defence
mechanisms by regulating
tyrosinaseactivity and
stimulating melanin production
Campillo MS, Gabaldon JA. 2008.
Rosmarinic acid, a photoprotective
agent against UV and
other ionizing radiations. Food
Chem. Toxicol. 47: 386-392.
5 Role of melanin
in skin
Melanin acts as free radical
scavenger in the skin
Herrling, T,Jung, K, Fuchs, J.
2007. The role of melanin as
protector against free radicals in
skin and its role as free radical
indicator in hair.
SpectroChemicaActa.A. 30: 1-7.
6 Antiinflammatory
Reduction of cyclooxygenase-
2 mRNAexpression,
prostaglandin E2 level and
ROS production were
Osakabe N, Yasuda A, Natsume M,
Yoshikawa T. 2003.
Carcinogenesis. Rosmarinic acid
inhibits epidermal inflammatory
7
significantly reduced by
pretreatment of animal with
Rosmarinic acid.
responses: anticarcinogenic effect
of Perillafrutescens extract in the
murine two-stage skin model. 25:
549-557
Till date there is no formulation available containing rosmarinic acid as sole photo-protectant.
Also, it has not been previously formulated to serve the function of decontamination. Thus, the
present work aims at formulating a suitable topicalsemi-solid formulation of rosmarinic acid for
effective photo-protection as well as radio-decontamination purposes.
BRIEF DESCRIPTION OF THE INVENTION
PROCEDURE
Prior to formulating semi-solid formulation of rosmarinic acid, it was evaluated for its preformulation
parameters such as solubility, melting point, IR analysis, partition coefficient etc. It
was also evaluated for anti-oxidant activity using TBARS (thiobarbituric acid reactive substance)
method and determination of toxicity using B16 Melanoma F1 cell lines was also done.
Preformulation was succeeded by formulation development and optimization. Optimization was
done on the basis of concentration of gelling agent, surfactant and pH modifier. Parameters such
as physical appearance, pH, viscosity and spreadability were considered for optimization.
TBARS method for estimation of antioxidant activity(Campilloet al., 2009)
Two grams of linoleic acid was solubilized in 50 ml ethanol. To it, 100 ml of 0.1 M phosphate
bufferpH 7.0 and 50 ml of water were added. Finally, the solution of rosmarinicacidand ascorbic
acid in 50 ml of ethanol was added for a final concentration of 200 mg/ kg. The reference
solution was prepared by final addition 50 ml of ethanol. Oxidation was performed by letting in
atmospheric air at 25ºC. For the detection of oxidative product formed, 5 ml of trichloro acetic
acid was added to 2.5 ml of oxidized solution for stopping the oxidation and then 2.5 ml of
thiobarbituric acid (0.67 % in water (w/v) was added. The reaction mixture was shaken and
incubated in a boiling water-bath for 15 minutes. The mixture was cooled and centrifuged at
1200 rpm for 20 min. The final colour developed was investigated at 532 nm using
spectrophotometer.
8
MTT Assay for toxicity evaluation:
The 3-(4, 5-dimethylthiazol-2-yl)-2, 5-diphenyltetrazolium (MTT) bromide dye reduction assay
was performed to determine the cytotoxic effect of rosmarinic acid at various concentrations.
Assay depends on the reduction of MTT by mitochondrial dehydrogenase, an enzyme present in
the mitochondria of viable cells, to a blue formazan product. Briefly, the B16 melanoma F1 cells
at a concentration 1 × 105 cells/ml were plated onto 96-well flat bottom culture plates with
various concentrations of rosmarinicacid.All cultures were incubated for 24 hours at 37 °C in a
humidified incubator. After 24 hours of incubation (37°C, 5% CO2 in a humid atmosphere), 5 μl
of MTT (5 mg/ml in DMSO) was added to each well, and the plate was incubated for a further
four hours at 37 °C. The resulting formazan was dissolved in 200 μl DMSO and absorbance of
the solution was measured at 595 nm using ELISA plate reader. All determinations were carried
out in triplicate. Concentrations of rosmarinic acid showing 50 % reduction in cell viability (i.e.
IC50 values) were calculated. General viability of cultured cells was determined through the
reduction of 3-(4,5-dimetylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) to formazan.
After rosmarinic acid treatment, cells were incubated for 48 hour at 37°C in a 5%CO2
atmosphere. MTT (1 mg/ml in PBS) was added to each well at a 1/10 volume of media. Cells
were incubated at 37°C for 3 hour, and DMSO was added in order to dissolve the formazan
crystals. The absorbance was measured at 570 nm using a spectrophotometer.
FORMULATION AND DEVELOPMENT
Cream was prepared by stirring method
Carbopol was added to a measured amount of water and stirred well. This mixture was allowed
to stand for a while so that carbopol gets swelled. Further, measured amount of water soluble
components such as rosmarinic acid, EDTA as a cross linking agent, PEG-400 as solubilizer and
triethanolamine as pH modifier were added to it for making up the water phase.
For oil phase, measured amount of sodium propyl paraben as preservative, stearic acid as
emulsifier, cetyl alcohol as emollient and cetostearyl alcohol as co-emulsifier were added.
9
Oil phase was added in water phase at 80 ºC with continuous stirring for 20-25 min and finally it
was homogenized till uniform emulsion is formed. To improve the aesthetic value of the product,
perfume was added in quantity sufficient. Table2 explains the detailed composition of optimized
formulation.
SR. NO
INGREDIENTS
(%)
CONCENTRATION (%)
1 Carbopol-940 0.475
2 Cetylalcohal 0.8
3 Cetostearylalcohal 0.1
4 Drug 0.018
5 Disodium EDTA 0.02
6 PEG-400 10
7 Sodium methyl paraben 0.3
8 Sodium propyl paraben 0.06
9 Stearic acid 2
10 Triethanolamine qs
11 Water (ml) upto 100 ml
Evaluation of optimized formulation:Optimized formulation was evaluated for pH, Viscosity,
spreadability, sun protection factor determination, in-vivo photo-protective studies and in-vivo
radio-decontamination studies.
Viscosity
Viscosity of creams was determined using a Brookfield Viscometer at 5, 10, 20, 30, 50 rpm
using spindle no.7 at 37 ºC.
10
Spreadability
The spreadability of creams was determined using wooden block glass slide apparatus (Madan et
al., 2010). It consists of a wooden block and provided with a pulley at one end. A rectangular
ground glass plate was fixed on this wooden block. Measured amount of cream under study was
placed on the ground glass plate, and thus cream was sandwiched between this plate and another
glass plate having same dimensions of ground glass plate attached with a hook. A weight of 300
grams was placed on the top of two plates for around 5 min so that cream spreads evenly
throughout the plate. Top plate was subjected to a pull of 30 grams with the help of a string
attached to the hook and the time (in second) required by top plate to cover a distance of 10 cm
was noted. Spreadability was calculated using mentioned formula:
Where, S=spreadability; m=weight tied to the upper glass slide; l=length of the glass slide and t
= time taken in seconds
Sun Protection Factor (SPF) determination (Dutra et al, 2004)
Ten grams of the sample was weighed and transferred to a 100 ml volumetric flask. It was
diluted to volume with ethanol, followed by ultrasonication for 5 min and filtered. A 5.0 ml
aliquot was transferred to 50 ml volumetric flask and diluted again to volume with ethanol. 5.0
ml of aliquot was further diluted up to 25 ml with ethanol. Absorption data was recorded at 5 nm
interval in the range of 290 to 320 using ethanol as a blank. Three determinations were made at
each interval point. The data was further used for calculating SPF by Mansur equation.
CF = 10 (Correction factor); EE (λ) = erythemogenic effect of radiation of wavelength; I (λ) =
intensity of solar light of wavelength; A (λ) = spectrophotometric value for absorbance of
wavelength.
In-vivo photo-protection studies
Male guinea pig weighing 500-600 grams were shaved and depilated 24 to 26 hours before
testing. All animal experimentations were carried out as per the approved protocol no.
S=ml/t
SPFspectrophotometric= CF ̽ Σ290
320 EE((λ) ̽ I(λ) ̽ Abs. (λ)
11
ISF/CPCSEA/IAEC/2010-46 by Institutional Animal Ethical Committee (IAEC) formed as per
the norms of Committee for prevention, Control and supervision of Experiments on Animals
(CPCSEA). The animals were divided into three groups. Group 1 was set as control; group 2 was
treated with 0.009 % rosmarinic acid containing cream and Group 3 was treated with 0.018
%rosmarinicacidcontaining cream. On the day of test, the animals were exposed to UV light for
30 min so that erythema starts appearing. Finally, preparations at different concentration of
rosmarinic acid were assayed by applying 1 gm of cream over a 1 × 1 cm area on side of dorsal
surface followed by checking the extent of erythema in the treated site after exposure to
ultraviolet rays. Reactions were read at 2, 4 and 12 hours interval post irradiation.
In-vivo radio-decontamination studies
Studies concerning in-vivo radio-decontamination were done at Institute of Nuclear Medicine
and Allied Sciences (INMAS), Defense Research and Development Organization (DRDO), New
Delhi. Animal used in the study were male wistar rats weighing from 150-200 grams.All animal
experimentations were carried out as per the approved protocol IAEC Wide no.-
INM/IAEC/2010/07/007 and as per the norms of Committee for prevention, Control and
supervision of Experiments on Animals (CPCSEA). Animals were shaved and depilated 24 to 26
hours before testing. At the time of test, the radioactive solution was applied at the exposed site.
The animals were exposed to this radioactive compound for different intervals of time.
Formulation containing different amount of rosmarinicacidwas applied on cotton puff and
exposed site was cleaned with help of this puff. Cream without rosmarinicacidwas used as
negative control and a solution of EDTA was used as positive control. The counts of radioactive
solution applied and cotton puff were measured using Capintec counter, USA. The results
obtained are expressed asPercentage efficacy = (counts from cotton puff/counts of radioactive
compound before application) × 100.
RESULTS AND DISCUSSION
Antioxidant Activity Determination
The antioxidant activity of Rosmarinic acid was studied using the TBARS test. Here the linoleic
peroxidation was indexed by measuring of oxidized product using TBARS test. Figure 1shows
the oxidation of linoleic with the time, measured by the formation of oxidized products at 532
12
nm, for different linoleic solutions when rosmarinicacidor ascorbic acid was added in a
concentration of 200 mg/kg at 25 ºC. Formation of oxidized product was much less in case of
rosmarinicacidas compared to standard ascorbic acid and control at the end of test. Thus, it
clearly indicates that amount of oxidized product was maximum in case of control and minimum
in presence of rosmarinic acid. This indicates that rosmarinic acid is better anti-oxidant as
compared to ascorbic acid.
Oxidized product formation measured at 532 nm versus time (days) for linoleic autooxidation
system
Figure 1 showing the results of TBARS test
Cell membrane consists of polyunsaturated fatty acids which are susceptible to auto-oxidative
peroxidation via free radical chain reaction. Free radicals include superoxide as well as hydroxyl
moiety along with singlet oxygen. Free radical scavenger such as rosmarinic acid can help
prevent such oxidation. TBARS method was done to evaluate antioxidant activity of rosmarinic
acid. Linoleicacid peroxidation was indexed by measuring of oxidized product production using
the TBARS test. Fig 1.shows the oxidation of linoleic with the time, measured by the production
of oxidized product at 532 nm, for different linoleic solutions when rosmarinic acid and ascorbic
acid was added in a concentration of 200 mg/kg at 25ºC. Production of oxidized product was
delayed the most when rosmarinic acid was used. This shows that rosmarinic acid has antioxidant
activity.Antioxidant activity of rosmarinic acid may be from the abstraction of hydrogen
atoms of the ortho-position hydroxyls on the rings A and B. This abstraction can occur
13
continuously to form a semiquinone structure, or even to form a quinone structure (Caoet al.,
2005). Structural analysis of rosmarinic acid reveals the presence of two catechol structure in
conjugation with a carboxylic acid. This accounts for the antioxidant activity of rosmarinic acid.
The antioxidant activity was even more than that of ascorbic acid(Del Bano et al., 2003).
MTT Assay
The effect of rosmarinicacidon viability of B16 melanoma F1 cells was checked using the MTT
assay. Rosmarinic acidreduced viability of B16 melanoma F1 in a dose-dependent manner, as
shown in Figure 2. After 24 hours of treatment, rosmarinicacidwas found to be cytotoxic to B16
melanoma F1 cells at concentrations of500 μg/ml and higher. Rosmarinic acidsolution at 500
μg/ml decreased the viability of B16 melanoma F1 cells to 50% of the initial level and thus it
was chosen as the IC50. Higher conc. of rosmarinicacidresulted in additional toxicity to the cells.
These results demonstrate that rosmarinic acid mediates a concentration and time dependent
increase in toxicity. As a result of these findings, 500 μg/ml concentration of rosmarinic acid was
found to be the IC50. Further experiments were carried out using same concentration to show the
effect of rosmarinic acid
Figure 2:Percent cell viability Vs Concentration of rosmarinic acid(μg/ml)
14
The 3-(4, 5-dimethylthiazol-2-yl)-2, 5-diphenyltetrazolium (MTT) bromide dye reduction assay
determines the cytotoxic effect of rosmarinic acid at various concentrations. It is a colorimetric
assay for measuring the activity of cellular enzymes that reduce the tetrazolium dye, MTT, to its
insoluble formazan crystal giving a purple color. Rapidly dividing cells have dehydrogenase
enzyme which reduces MTT to its insoluble formazan crystal. More the drug is cytotoxic, lesser
will it contain the enzyme and lesser will the colored product formed. Absorbance of such
solution helps give direct indication of viability of cell and the associated cytotoxicity exhibited
at such a concentration. Results of MTT assay on B16 melanoma F1 cell lines shows that
rosmarinic acid is non-toxic at low dose (up to500 μg/ml) but toxicity increases as the dose of
rosmarinic acid is increased.
EVALUATION PARAMETERS OF PREPARED CREAM
All the formulations were evaluated for all physical evaluation parameters such as appearance,
pH, viscosity, spreadability and sun protection factor (SPF).
Table 3: Comparison of physical parameters of developed formulations
Sr. No Parameter Result
1 Appearance Slightly yellow
2 pH 6.2
3 Viscosity 3370 ± 150 at 10 rpm with spindle no. 7
4 Spreadability 9.7 gm.cm/sec.
5 Sun protection factor 1.28
The developed formulations were slightly yellowish in colour. Results of pHsuggested that
formulation is compatible with skin. Viscosity result shows that formulation has satisfactory
flow property and has good consistency.The spreadability of developed formulations was
calculated and obtained results are explained in Table 3. The results show that the developed
formulations have good spreadability and are within the acceptable range. It forms a smooth film
on the surface of skin.The values of SPF were calculated and reported. These values suggest that
the results approach towards photo-protection activity.
15
The optimization was done on the basis of polymer, surfactant as well as pH. After optimization,
it was found that amongst all the developed formulations, formulation containing 0.018% of drug
was found to be most suitable formulation. It was found to be slightly yellowish in appearance.
The pH of optimized formulation was 6.2, which was found to be compatible with pH of the
skin. Spreadability of optimized formulation was 9.7 which facilitate an even layer on the skin.
SPF of optimized formulation was 1.28 which approach towards effective sun protection.
According to USFDA press release, 2011, SPF value focuses on UVB-related burns (erythema
and redness) and negates UV-A related premature aging component.Therefore, a high SPF
sunscreen does not necessarily guarantee effective photo-protector in full UV spectrum.Photoprotective
agents who can concomitantly absorb UV as well as prevent inflammation mediated
by free radicals can serve as effective photo-protectors in full UV spectrum. Absorbance of
formulation was taken at definite interval from 280-320 nm (UV-B) for calculation of SPF value.
The results were summed and calculated value came to be 1.28. Thus, rosmarinic acid emerges
as eligible candidate which exhibit absorption in UV-B range as well as serves as free radical
scavenger as discussed above.
In-vivo photoprotection studies
In invivophotoprotection studies, all the three groups were first treated with cream (with or
without containing rosmarinic acid) and UV rays were allowed fall. Results were expressed as
prevention of erythema after 2, 4, and 12 hours of application of cream. In this study guinea pigs
were divided in three groups. All the results are explained in Figure3. First group was treated
with cream without romarinic acid, second group treated with 0.009 % cream containing
rosmarinic acid and third group treated with cream containing 0.018 % rosmarinic acid. It is
clearly observed from the Figures that cream with higher concentration of rosmarinic acid(0.018
%) have better photoprotection activity. This is confirmed by marked prevention of erythema
after exposure of UV rays to the animal.
Figure 3:
Effect of UV rays after application of cream without rosmarinic acid(first group)
16
A) At 2 hours after exposure of UV rays (B) At 4 hours after exposure of UV rays
(C) At 12 hours after exposure of UV rays
Effect of UV rays after application of formulation with 0.009% rosmarinic acid(second
group)
A) At 2 hours after exposure of UV rays (B) At 4 hours after exposure of UV rays
17
(C) At 12 hours after exposure of UV rays
Effect of UV rays after application of formulation – with 0.018 % rosmarinic acid(third
group)
(A) At 2 hours after exposure of UV rays (B) At 4 hours after exposure of UV rays
(C) At 12 hours after exposure of UV rays
18
Invivophoto-protection studies proved that rosmarinic acid is photo-protective at dose of 0.018
%. Results were encouraging for cream containing 0.009% of rosmarinic acid. However, the
cream with 0.018% rosmarinic acid showed marked decrease in erythema and edema at 2hrs,
4hours and 12 hours post radiation.At 2 hours post irradiation, a slight erythema was perceptible
which faded at 12 hours with cream containing 0.018% rosmarinic acid.
In-vivo radio decontamination studies
In the radio decontamination study counts before and after application of cream was determined.
Counts were used to determine percentage efficacy of decontamination byrosmarinicacidin
comparison to standard EDTA. Radio decontamination studies proved that formulation
containing rosmarinicacidhave satisfactory radio decontaminating efficacy. Figure 4shows the
treated animals for radio decontamination study.
Figure 4: Animals treated for radio decontamination study
19
Table 4 show the comparison of efficacy of cream containing rosmarinic acid at different
concentration with that of different concentration of EDTA at different interval of time.
Figure 5: A graph showing decrease in efficacy of cream containing rosmarinic acid and EDTA
solution with time. X-axis shows the time in minutes while Y-axis shows % efficacy of
formulation.
Time
(Mins)
F1
(0.018
% RA)
F2
(0.09%
RA)
F3
(0.18%
RA)
F4(1%
EDTA)
F5(2%
EDTA)
F6 (5%
EDTA)
F7(7%
EDTA)
10 29.8 56.4 73.9 38.05 43.42 68.9 78.5
20 27.06 40.25 72.6 36.17 38.2 52.5 68.3
30 26.05 35.75 65.9 30.05 36.71 41.8 50.6
20
From in-vivo radio decontamination studies it is observed that low concentration of rosmarinic
acid gives the same decontamination efficacy as is given by high concentrations of EDTA which
is positive control. Results shows that cream containing rosmarinic acid at 0.18% shows an effect
equivalent to that obtained for 7% EDTA. The observed decontamination efficacy was 73.9% at
10 mins post exposure. For 20 and 30 minutes post exposure, decontamination efficacy was
observed to be 72.6% and 65.9% respectively for 0.018%. A similar decrease in decontamination
efficacy was observed for cream with 0.09% rosmarinic acid where decontamination efficacy
measured at 10, 20, 30 minutes was 56.4%, 40.25% and 35.75% respectively. Experiments as
positive control were done taking solution of EDTA at 1%, 2%, 5% and 7%. Cream at three
varying concentration of rosmarinic acid at 0.018%, 0.09% and 0.18% was studied. It was
evidenced from the results that a decrease in radio-decontamination efficacy was observed for
both EDTA as well as for rosmarinic acid. However, the decrease is more pronounced in case of
EDTA as compared to that for rosmarinic acid containing formulation.
This study indicated thatoptimized formulation have good decontamination efficacy. The
probable reason for such activity may be attributed to the presence of multiple –COOH as well as
–OH bonds which act as effective binding site for the metal thus facilitating its removal. These
group acts as binding center for the metal.
Skin irritation study proved that rosmarinic acid is non-toxic and non-irritant to the skin. Thus,
all the results particularly SPF value, photo-protection study, anti-oxidant study, skin irritation
study and radio decontamination study suggests that developed and optimized cream formulation
is the best suitable formulation for sunscreen as well as radio-decontamination purpose. It is safe,
effective and stable.
References:
1. ArmeniT, DamianiE, Battino M,Greci L, Principato G. 2004. Lack of in vitro protection by a
common sunscreen ingredient on UVA-induced cytotoxicity in keratinocytes. Toxicology.
203 (1–3): 165–178.
2. Knowland J,McKenzieEA,McHughPJ,Cridland NA.,1993. Sunlight-induced mutagenicity of
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We Claim:
1. A method of Preparation of a formulation for its Photoprotective and Radio decontamination
activity comprising the steps of:
adding carbopol in a measured amount of water and stirred well and is allowed to stand for a
while so that carbopol gets swelled;
adding measured amount of water soluble components such as rosmarinic acid, EDTA as a cross
linking agent, PEG-400 as solubilizer and triethanolamine as pH modifier for maling up water
phase;
adding measured amount of sodium propyl paraben as preservative, stearic acid as emulsifier,
cetyl alcohol as emollient and cetostearyl alcohol as co-emulsifier so as to obtained oil phase;
adding Oil phase in water phase at 80 ºC with continuous stirring for 20-25 min and finally it is
homogenized till uniform emulsion is formed.
2. A pharmaceutically formulation comprises the
Carbopol-940 (0.475%), Cetylalcohal (0.8%), Cetostearylalcohal (0.1%), Drug (0.018%),
disodium EDTA (0.02%), PEG-400 (10%), Sodium methyl paraben (0.3%), Sodium propyl
paraben (0.06%), Stearic acid (2%), Triethanolamine and water to make vloume 100 ml.
3. The formulation as claimed in claim 2, wherein viscosity of said formulation is determined
using a Brookfield Viscometer at 5, 10, 20, 30, 50 rpm using spindle no.7 at 37 ºC.
4. The formulation as claimed in claim 1,wherein antioxidant activity of Rosmarinic acid is
studied using the TBARS test.
5. The formulation as claimed in claim 2,wherein linoleic peroxidation is indexed by measuring
of oxidized product using TBARS test.
6. The formulation as claimed in claim 2,wherein oxidation of linoleic with the time, measured
by the formation of oxidized products at 532 nm, for different linoleic solutions when
rosmarinicacidor ascorbic acid was added in a concentration of 200 mg/kg at 25 ºC.
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7. The formulation as claimed in claim 2,wherein Formation of oxidized product is much less in
case of rosmarinicacidas compared to standard ascorbic acid and control at the end of test.
8. The formulation as claimed in claim 2,wherein amount of oxidized product ismaximum in
case of control and minimum in presence of rosmarinic acid.
9. The formulation as claimed in claim 2 to claim 8, wherein said formulation is used for
Evaluation and Characterization for its Photoprotective and Radio decontamination activity.