Field of the Invention
The present invention relates to a skin whitening composition comprising the whole
plant extract of Coldenia procumbens. Particularly the present invention relates to the
alcohol extracts of whole plant of Coldenia procumbens. More particularly the
composition of the present invention provides promising skin whitening property
(tyrosinase inhibition).
Background and Prior art of the Invention
Coldenia procumbens Linn (Doraginaceae) is a procumbent, deep rooted, hairy herb,
found throughout India as a weed in moist places. The powdered roots are administered
in leucorrhoea and menorrhagia. The leaves are taken internally to improve the
resistance power. The leaves are also used as a diuretic.
The medicinal plant Coldenia procumbens is more extensively used as a folk remedy in
a number of disease conditions and there has been long-term usage of this plant in
ayurveda. The scientific literature on Coldenia procumbens is very scarce and no patent
has been awarded till now for any work involving this plant.
Lenin Bapuji and Venkat Ratnam conducted a review on traditional uses of some
medicinal plants by tribals of Gangaraju Madugula mandals of Visakhapatnam District,
Andhrapradesh (Ethnobotanical leaflets, 13: 388-98, 2009). The review reported that the
leaf of Coldenia procumbens is applied as a remedy for rheumatic swellings.
The in-vivo anti-inflammatory activity has been reported for the ethanolic extract of the
aerial parts of Coldenia procumbens. (Arun et al; Pakistan Journal of Pharmaceutical
sciences, Vol 18, No.3, pp. 17-20 (2005) The results confirm that the ethanolic extract
of Coldenia procumbens produced significant anti-inflammatory activity when
compared to the standard Indomethacin.
However, no studies have been reported either in the scientific literature or patent
domain on the skin whitening or anti-aging properties of Coldenia procumbens.

Some of the skin care formulations currently available in the market containing herbal
extracts have a variety of beneficial effects to the skin like skin whitening, anti-aging
and anti-wrinkling.
The present inventors have found that skin care formulation comprising the herbal
extract of Coldenia procumbens provide novel skin-whitening property by suppressing
the production of melanin in the skin.
Object of the invention
It is an object of the present invention to provide a skin cream composition containing
the herbal extract of Coldenia procumbens exhibiting the skin lightening effect.
Summary of the invention
According to one aspect of the present invention, there is provided a herbal composition
comprising extracts of Coldenia procumbens in amount of 0.5 - 10% for skin
lightening.
According to another aspect of the present invention there is provided a process for
preparation of herbal formulation comprising whole plant extract from Coldenia
procumbens comprising steps of hot mixing, cmulsification followed by cooling.
Brief Description of Accompanying Figures
Fig 1: Percent inhibition of tyrosinase enzyme by few herbal extracts.
Fig 2A: Percent inhibition of tyrosinase enzyme by various solvent extracts of whole
plant (Aqueous, F.thanol & Petroleum ether) of Coldenia procumbens.
Fig 2 B: Percent inhibition of tyrosinase enzyme by whole plant of Coldenia
procumbens at varying concentration.

FIG 2 C: DPPH radical scavenging potential of Coldcnia procumbcns at varying
concentration.
FIG 2 D: Inhibition of linolcic acid oxidation by various solvent extracts (Aqueous.
Ethanol & Petroleum ether) of Coldenia procumbens whole plant.
FIG 3: Tyrosinase inhibition assay of skin creams comprising extracts from Coldcnia
procumbens at various concentrations.
Detailed Description of the Invention
The color of mammalian skin and hair is determined by a number of factors, the most
important of which is the degree and distribution of melanin pigment. Melanin is
synthesized through melanogenesis by the key enzyme tyrosinase in a multistep
biochemical process. Skin darkening is due to the hyper-secretion of melanin from
melanocytes. So, the compounds exhibiting melanin synthesis inhibitory activity are the
potential agents in skin lightening. Thus, in recent years, cosmetic compositions have
been developed to reduce the amount of melanin in the skin and therefore, whiten the
skin.
In accordance with the present invention, a skin care formulation comprising the herbal
extract from the whole plant of Coldenia procumbens inhibits the tyrosinase enzyme.
The active ingredients from the above said extract incorporated in skin care
formulations hardly cause adverse effects, as it is commonly used in ayurvedic
preparations for oral as well as for topical application.
The herbal skin care formulation of the present invention comprises aqueous or ethanol
or petroleum ether extract of Coldenia procumbens whole plant.
The skin care formulation of the present invention comprises ethanolic extract of
Coldcnia procumbens whole plant wherein said extract is present in an amount of 0.5 to
10.0 % by weight, preferably 0.5 to 5% by weight, most preferably 1 to 3% by weight.
The formulation further comprises of conventional carrier and additives.

Although the exact mechanism of tyrosinase inhibition is not known, it is believed that
the above said extract complexes with or chelates the copper present in the active site of
tyrsoniase enzyme and thus inhibits the synthesis of melanin.
The present invention also provides preparations that include the skin whitening
compositions. The composition may be prepared in various forms. For example, it may
be in the form of a cream, gel, lotion, powder, or as an emulsion or ointment. In each
formulation, various known conventional cosmetic ingredients may be incorporated. For
example, cosmetic ingredients like alcohols, fats, and oils, surfactants, fatty acids,
silicone oils, humectants, moisturizers, viscosity modifiers, emulsifiers, stabilizers,
coloring agents, and perfumes may be included.
Other components that may be included in the composition include sunscreen, octyl
methyl cinnamate.
The carriers include polyoxyethylenc (2) stearyl ether, polyethylene (21) stearyl ether
and isopropyl palmitate.
Further conventional additives include stearic acid, cetostearyl alcohol, mineral oil and
propylene glycol.
The process for preparation of herbal formulation comprising extract from Coldenia
procumbens whole plant comprising steps of hot mixing, emulsification followed by
cooling.
The oil phase ingredients like polyethylene (21) stearyl ether, Isopropyl palmitate,
Stearic acid, Cetostearyl alcohol, Mineral oil and Glyceryl monostearatc SE were mixed
together. This mixture - I was heated till the temperature of the mixture reaches 75-
80°C. In the same manner, the water phase ingredients like Polyoxyethylenc (2) stearyl
ether; Disodium EDTA, Sodium meta bisulphatc and distilled water were mixed
together. This mixture - II was also heated separately till the temperature of the mixture
- II reaches 75-80°C. Once the temperature of two mixtures II and I reaches 75-80°C,
they were mixed together and homogenized for 30 minutes under constant stirring. This
process is called emulsification. Once the temperature of the mixture reaches 55-58°C,
the preservatives like Methyl/Propyl paraben and 2-Phenoxyethanol were added. Once
the temperature of the mixture drops down to 40°C, the clhanolic extract from Coldenia

procumbens was added to the formulation. The desired fragrance for the formulation is
added at 35 - 38 °C. All additions were carried out under constant stirring.
The composition of the present invention is particularly well suited for topical
application and for use as skin whitening agent.
The present invention also provides a method for determining an acceptable extract of a
plant for use in skin care compositions. The skin lightening effect of the herbal extract
incorporated into skin care formulation was examined by its tyrosinase inhibiting
activity using mushroom tyrosinase as the enzyme source.
Studies have also been performed in various other herbal plants for skin
whitening/tyrosinase inhibitory activity. From the in vitro screening, it has been found
that ethanolic extract of Coldcnia procumbens exhibited the maximum tyrosinase
inhibitory effect when compared to all other herbals. The efficacy of the herbals for
tyrosinase enzyme inhibition was evaluated and was expressed in terms of % inhibition.
Detailed Description of the Accompanying figures
FIG 1 represents the tyrosinase inhibitory activity of various herbals. From the findings,
it is evident that the ethanolic extract of Coldenia procumbens exhibited maximum
tyrosinase inhibiting property than any other herbals studied in the current work.
FIG 2A illustrates percent inhibition of tyrosinase enzyme by various solvent extracts of
whole plant (Aqueous, Ethanol and Petroleum ether) of Coldenia procumbens. From the
studies, it was found that ethanolic extract of Coldenia procumbens showed better
inhibition of tyrosinase enzyme than other solvents.
FIG 2B indicates the percent inhibition of tyrosinase enzyme by whole plant of
Coldenia procumbens at varying concentrations. The results revealed that the tyrosinase
inhibitory activity of ethanolic extract of Coldenia procumbens whole plant was
elevated in a dose dependant manner.

FIG 2C represents the DPPH radical scavenging potential of Coldenia procumbens at
varying concentration. The data infers that the scavenging of DPPH radical incremented
with increasing concentration of the plant extract.
FIG 2 D elucidates the inhibition of linoleic acid oxidation by various solvent extracts
(Aqueous, Ethanol and Petroleum ether) of Coldenia procumbens whole plant. The
linoleic acid was used as the model system for evaluating the antioxidant potential of the
plant extracts. From the studies, it is noted that cthanolic extract of Coldenia
procumbens is superior in inhibiting linoleic acid oxidation than any other solvent used.
FIG 3 illustrates the tyrosinase inhibition assay of skin creams comprising extracts from
Coldenia procumbens at various concentrations. The cream formulation comprising
1%, 2%, 3% and 5% of extract by weight shows dose dependant inhibition of tyrosinase
enzyme and it attains saturation at 3% level.
The present invention is illustrated by the following non-limiting examples. It is to be
understood that the disclosed methodology is not limited to the exact details briefed here
and variations to implement the idea are possible. The methodology described is for the
purpose of description and should not be taken as limitation.
Example 1
Extracts from Coldenia procumbens in the present invention was prepared by extracting
whole plant of Coldenia procumbens in water, alcohol and Petroleum ether (1:10 ratio)
and continuously extracting for 1hr at 50°C. The mixture was cooled to room
temperature, filtered and centrifuged the filtrate to obtain green liquid and compared
with known compositions.
The skin beneficial properties like skin lightening exhibited by Coldenia extract were
evaluated by several standard in-vilro biochemical assays, (e.g. Tyrosinase inhibition
assay for skin whitening).
Example 2
Mushroom tyrosinase inhibition assay was carried out by incubating the enzyme with
buffered substrate with and without inhibitors for 30 mins. The absorbance was read at

492 nm and the percentage inhibition of the extracts on the enzyme was calculated
(Kang Tae Lee, Kwang Sik Lee, Ji Hean Jeong. Byoung Kee Jo, Moon Young Heo, and
Hyun Pyo Kim, 2003. Inhibitory effects of Iamul'us mori extracts on melanogenesis. /.
Cosmet. sci., 54, 133-142). The results were compared with various herbal extracts.
The percent inhibition of tyrosinase activity by various herbal extracts is given in Table
1. It is quite evident from these data that ethanolic extract of Coldcnia procumbcns is
superior in its tyrosinase inhibitory activity than any other plant extracts used in the
current studies.
Table 1. Percent inhibition of tyrosinase by herbal extracts

Note: ET-Ethanolic extract
Example 3:
Table 2 provides a skin cream formulation containing extracts of Coldenia procumbens.
The herbal extract was prepared with alcohol from the whole plant of Coldcnia
procumbens. The oil phase ingredients like polyethylene (21) stearyl ether, Isopropyl
palmitate, Stearic acid, Cetostearyl alcohol, Mineral oil and Glyceryl monoslearate SE
were weighed separately and mixed together. This mixture - I was heated till the
temperature of the mixture reaches 75-80°C. In the same manner, the water phase
ingredients like Polyoxyethylene (2) stearyl ether; Disodium EDTA, Sodium meta
bisulphate and distilled water were weighed separately and mixed together. This mixture

- II was also heated separately till the temperature of the mixture - II reaches 75-80 °C.
Once the temperature of two mixtures reaches 75-80 ° C, they were mixed together and
homogenized for 30 minutes under constant stirring. Once the temperature of the
mixture reaches 55-58°C, the preservatives like Methyl/Propyl paraben and 2-
Phenoxyethanol were added. Once the temperature of the mixture drops down to 40 ° C,
the extract from whole plant of Coldenia proewnbens was added to the formulation. The
desired fragrance for the formulation is added at 35 - 38°C. All additions were carried
out under constant stirring.
Table 2. Skin cream formulation containing extract from Coldenia
procumbens.

Example 4:
The free radical mediated damage to the cellular macromolecules will results in aging.
The way of addressing the free radical mediated aging is to protect the cellular
macromolecules either by providing them with the assistance of defense system which
repairs and/or counteracts the reactive free radicals. One such mode of screening the
potent antioxidants for their counteracting ability is by the DPPH radical scavenging
assay. The DPPH radical is a synthetic free radical, which is easily available from the

commercial source and is quiet stable when compared to the hydroxy or nitroxy
radicals. Hence it has been opted (widely accepted) as a model system for screening the
antioxidant activity of any molecule.
DPPH radical scavenging assay was performed by mixing DPPH solution with and
without plant extract in a microtitre plate. The reaction mixture was incubated at 25°C
for 5 minutes, after that the absorbance was measured at 520 nm. The DPPH radical
scavenging activity of the plant extract was calculated as the percentage inhibition. The
DPPH scavenging activity of plant extracts at varying concentration was depicted in
Figure 2 C. The IC 50 has been computed from Fig 2C and the value is 80 mg/ml.
Example 5:
Linoleic acid was used as the model system for evaluating the antioxidant potential of
plant extracts. A mixture containing phosphate buffer (0.05 M, pH 7.0), water, plant
extract and linoleic acid in absolute ethanol (2.5%) was placed in a vial with a screw cap
and then placed in a dark oven at 40 °C for overnight. To this incubation mixture, added
ethanol (75%) and ferrous chloride (0.02M) in 3.5% T1C1. Add ammonium thiocyanate
(30%), precisely 3 minutes after the addition of ferrous chloride. The absorbance of the
red colour was measured at 500 nm every 24 hour until the absorbance of the control
reached it's maximum. The antioxidant capacity of plant extracts on inhibition of
linoleic acid oxidation was presented in Figure 2 D

We claim
1. A herbal composition comprising extract of Coldenia procumbens in amount of
0.5 -10% for skin lightening.
2. The composition as claimed in claim 1, wherein the extract comprises the whole
plant of Coldenia procumbens.
3. The herbal composition as claimed in claim 1 wherein the said extract is present
in an amount of 0.5 to 5.0% by weight.
4. The herbal formulation as claimed in claim 3 wherein said solvent is selected
from water, ethanol and petroleum ether.
5. The herbal formulation as claimed in any one of preceding claim further
comprises conventional additives selected from the ethers, fatty acids and fatty
alcohols.
6. The herbal formulation as claimed in claim 5 wherein the said ethers is selected
from emulsifiers namely polyoxyethylene lauryl ether, polyoxyethylene alkyl
ether, polyoxyethylene octyldodecyl ether, polyoxyethylene cetyl ether,
polyoxyethylene stearyl ether, polyoxyethylene oleyl ether, polyoxyethylene
lauryl ether, polyoxyethylene myristyl ether, and polyethylenestearyl ether.
7. The herbal formulation as claimed in claim 5 wherein the said fatty acids is
selected from lauric acid, myristic acid, palmitic acid, stearic acid, arachidic
acid, linoleic acid, alpha linolenic acid, docosahexanoic acid, oleic acid,
isopropylpamitic acid, glyceryl monostearic acid.
8. The herbal formulation as claimed in claim 5 wherein the said fatty alcohols is
selected from capric alcohol, lauryl alcohol, myristyl alcohol, celyl alcohol,
palmitoyl alcohol, isostearyl alcohol, cetostearyl alcohol, and linolcyl alcohol
along with mineral oil and propylene glycol.

9. A process for preparation of herbal formulation comprising whole plant extract
from Coldenia procumbens comprising steps of hot mixing, emulsification
followed by cooling.
10. The process for preparation of a herbal formulation as claimed in claim 6
wherein the extraction from whole plant involve heating the powdered solid of
the whole plant with solvent, followed by filtration, cooling and centrifugation.
11. The process for preparation of an herbal formulation as said in claim 7, wherein
the solvent is selected from water, ethanol and petroleum ether.
12. The composition of claim 1 adapted to act as tyrosinase inhibitors or free radical
scavengers or both.
13. A herbal composition comprising extract of Coldenia procumbens and a process
of its preparation as described herein with reference to figures and examples.

Herbal skin care formulations comprising the extract of Coldenia procumbens exhibiting
the skin lightening property and a process for preparation of the same. The present
invention further provides skin care formulations comprising the extract of Coldenia
procumbens resulting in inhibition of tyrosinase enzyme, thereby providing skin
whitening property and a process for preparation of same.