Field of the invention
The present invention relates to herbal formulation derived from the extracts of leaves, bark or
fruits of Ziziphus mauritiana, useful for protection of skin from ageing. More particularly, the
formulation of the present invention comprising extract from the said plant provide superior skin
benefits in terms of skin whitening (tyrosinase inhibition) and anti-aging (anti-oxidant property).
Background and prior art
A large number of skin care formulations are available for addressing the skin benefits like
whitening, anti-wrinkle, anti-aging, skin tightening and UV protection. These formulations when
topically applied on the skin may or may not interfere in the melanin synthesis, a key
characteristic of skin lightening. The key actives added in these formulations may be of natural
origin or synthetic compounds, therefore posing minor or significant side effects.
Ziziphus mauritiana is a spiny, evergreen tree up to 15m high, with trunk 40 cm or more in
diameter. The fruit is of variable shape and size. When slightly under-ripe, this fruit is a bit juicy
and has a pleasant aroma. Ziziphus mauritiana is grown in different parts of India and is known
for its effects on various medicinal properties. The roots of Ziziphus species are commonly used
in folklore medicine for the treatment of diarrhea, digestive disorders, weakness, liver
complaints, obesity, urinary troubles and skin infections.
The fruits are applied on cuts and ulcers for cure. Mixed with oil, they are rubbed on rheumatic
areas for pain relief. The leaves are applied as poultices. The bitter, astringent bark decoction is
taken to halt diarrhoea and dysentery and relieve gingivitis. The bark paste is applied on sores.
An infusion of the flowers serves as an eye lotion.
Due to the widely usage of various parts of Ziziphus mauritiana in various medicinal
preparations and the scarcity of its application in any cosmetic field, a study was undertaken to
explore the skin benefits of this plant. Hardly any scientific reference describing the use of
Ziziphus mauritiana species in a skin cream formulation exhibiting the tyrosinase inhibition
properties is known.

A study was conducted by Ruy J. V. Alves ( J. Braz. Chem. Soc, Vol. 16, No. 3B, 654-656,
2005.), wherein they have identified the chemical components of the Ziziphus mauritiana fruit.
The sugars identified to be present in Ziziphus mauritiana were galactose, fructose and glucose.
The two phenolic compounds identified by TLC were caffeic acid and p-coumaric acid. Phenolic
compounds were also quantified using HPLC. Twelve peaks of phenolic compounds were
detected. Among these, p-hydroxybenzoic acid, caffeic, ferulic acid and p-coumaric acid were
the most abundant, whereas vanillic acid was the least abundant.
In a published research paper (Mahesh and Satish, 2008, World Journal of Agricultural Sciences
4 (S): 839-843), on the antimicrobial activity of some important medicinal plant against plant
and human pathogens, there was an experimental observation that the barks and leaves of
Ziziphus mauritiana have excellent anti-microbial properties.
Objects of the invention
The primary objective of the invention is to provide a skin composition comprising extract of
Ziziphus mauritiana in amount of 0.5-5% for the skin whitening activity.
A further objective is to provide a composition comprising extract of Ziziphus mauritiana in
amount of 0.5-5.0% fox the anti-oxidant activity, which signifies the anti-aging property.
Summary of the invention
According to one aspect of the present invention there is provided a herbal formulation
comprising extracts of Ziziphus mauritiana leaves, bark and fruits in solvent selected from water,
alcohol and petroleum ether.
According to another aspect of the present invention there is provided a process for preparation
of a herbal composition comprising extract of Ziziphus mauritiana - leaves/bark/fruits
comprising steps of hot mixing, emulsification followed by cooling.

Brief Description of the Accompanying Figures
Fig 1 A: Percent inhibition of tyrosinase enzyme by few herbal extracts.
Fig 1 B: Percent inhibition of tyrosinase enzyme by various parts (Leaf, Bark & Fruit) of
Ziziphus mauritiana.
Fig 1 C: Percent inhibition of tyrosinase enzyme by various solvent extracts (Aqueous, Ethanol
& Petroleum ether) of Ziziphus mauritiana leaves.
Fig 1 D: Percent inhibition of tyrosinase enzyme by Ziziphus mauritiana leaves at varying
concentration.
Fig 2 A: Percent inhibition of LPO by few herbal extracts.
Fig 2 B: Percent inhibition of LPO by extracts from various parts (Leaf, Bark & Fruit)
of Ziziphus mauritiana.
Fig 2 C: Percent inhibition of LPO by various solvent extracts (Aqueous, Ethanol & Petroleum
ether) of Ziziphus mauritiana leaves.
Fig 2 D: Percent inhibition of scavenging DPPH radical by Ziziphus mauritiana leaves at varying
concentration.
Detailed Description of the Invention
The properties of Ziziphus mauritiana as a remedy for various ailments are known in the
traditional medical literature and its practice by humans put this herb in the list of potential safe
ingredients. Moreover, the fruits Ziziphus mauritiana is regularly consumed by humans. With
this range of usage by human beings, it can be readily used in product formulations for topical
application on human skin.
It has been established that Ziziphus mauritiana to provide superior skin benefits in terms of skin
whitening (tyrosinase inhibition) and anti-aging (anti-oxidant property). The extract has no or
low side effects when compared with other chemical ingredients, as they are known in traditional
medical practices.

A group of enzymes namely, tyrosinase and tyrosinase related proteins are responsible for skin
darkening as they are involved in melanogenesis. Inhibition of these enzymes prevents the
melanin accumulation, responsible for the skin darkness.
The present invention is directed towards formulation comprising herbal extracts of Ziziphus
mauritiana leaves or bark or fruit to inhibit the tyrosinase enzyme, to protect the skin from aging
through the anti-oxidant property. The composition comprises extract of Ziziphus mauritiana
leaves, bark or fruit in alcohol or petroleum ether.
The chemical components present in the alcohol extract are responsible for the tyrosinase
inhibition properties. Similarly the chemical components present in the aqueous extracts are
responsible for the antioxidant activity.
The composition of the skin cream in the present invention comprises alcohol extracts of
Ziziphus mauritiana leaves in amount of 0.5 tolO.O %, preferably 0.5 to 5%, most preferably 1 to
3%. The details of the concentrations of other ingredients are given in the Table 1 below:
Table 1. Skin cream formulation containing Ziziphus mauritiana extract



Other components that may be included in the composition include sunscreen, Octyl methyl
cinnamate.
The carrier include polyoxyethylene (2) strearyl ether, polyethylene (21) stearyl ether and
isopropyl palmitate.
Further conventional additives include stearic acid, cetostearyl alcohol, mineral oil and
propylene glycol.
The process of preparing the composition comprises the steps of hot mixing and emulsification
followed by cooling.
The invention is now defined by way of non limiting illustrative examples:
Example 1.
A composition comprising Ziziphus mauritiana as in the present invention was prepared by
extracting dry leaves in water/alcohol/Petroleum ether (1:10 ratio) and continuously extracting
for lhr at 50°C. The mixture was cooled to room temperature, filtered and centrifuged the filtrate
to obtain yellowish green liquid and compared with known compositions.

The skin beneficial properties exhibited by Ziziphus mauritiana extracts, like skin lightening and
anti-aging were evaluated by several standard in-vitro biochemical assays, (e.g. Tyrosinase
inhibition assay for skin whitening, Lipid peroxidation inhibition assay and DPPH radical
scavenging assay for the anti-oxidant activity.)
A number of herbal extracts in the current studies showed significant activities beneficial to the
skin. Herbal extracts derived from Ziziphus mauritiana leaf extracts showed skin lightening
properties superior to the well-known Glycyrrhiza glabra (licorice) extract and others used in the
study. Skin darkening/toning is due to the production of a group of pigments called Melanin. The
biochemical pathway involved in the production of melanin is a multi-step process, catalyzed by
various enzymes. Tyrosinase is the key regulatory enzyme involved in initiating the pigment
production. If an inhibitor either from natural or synthetic source blocks the activity of this
enzyme, it will result in the skin lightening benefits. In order to screen the herbals for its skin
lightening property, tyrosinase enzyme isolated from the mushroom was used as a model system
in the cosmetic industry over decades. From the in-vitro experiments, it is ascertained that the
extract of Ziziphus mauritiana leaves contains compounds capable of blocking/suppressing one
or more steps in the biochemical pathways of melanin synthesis.
During the current work, we evaluated the efficacy of the plant extract in inhibiting the activity
of enzyme and it was expressed in terms of percentage inhibition (in comparison with the
corresponding control). The percentage inhibition of tyrosinase by Ziziphus mauritiana was
much higher than that exhibited by licorice extracts of similar concentrations; the latter extract is
commonly used in commercial formulations of skin lightening creams. These superior multi-
beneficial properties of Ziziphus mauritiana open up potential opportunities for its usage in skin
creams as skin whitening and anti-aging actives.
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 famulus mori extracts on melanogenesis. / Cosmet. sci., 54, 133-142). The results
were compared with various herbal extracts and also the Glycyrrhiza glabra extract, which is
normally used in commercial skin whitening creams, acting through its tyrosinase inhibition

roperty. The percent inhibition of Ziziphus mauritiana was found to be superior to Glycyrrhiza
xtract.
he percent inhibition of tyrosinase activity by various herbal extracts is given in Table 2. It is
uite evident from these data that Ziziphus mauritiana leaf alcohol extract is superior in its
'rosinase inhibition activity and better than a commercially used skin whitening licorice extract.
Table 2. Percent inhibition of tyrosinase by herbal extracts

Note: AQ -Aqueous extract, PE- Petroleum Ether extract,
ET- Ethanol extract, ME- Methanol extract

Example 2
In the process of aging, there is an imbalance between the reactive oxygen/ nitrogen species and
its counteracting antioxidant systems in the cell. Such imbalance results in the free radical
mediated damage of the cellular macromolecules. Membrane lipids are the major site of attack
for the free radicals because cell membrane comprises of the unsaturated fatty acids like linoleic
acid, linolenic acid and Arachidonic acid. Since unsaturated fatty acids easily prone to free
radical mediated damage and results in the formation of lipid peroxides. .Thus free radical
mediated damage to lipids results in the altered fluidity of cell, which reflects in the texture of
skin.
One-way of protecting the skin texture due to aging is to prevent the lipid peroxidation. Such
prevention can happen in the aging skin though the supplement of antioxidants to the skin cell
through topical applications. Since medicinal plants are the very good source of antioxidants,
they are evaluated for their antioxidant potential by using linoleic acid micelles as a model of
membrane.
In the standard protocol used, a predetermined amount of linoleic acid was oxidized by air at 40
°C by overnight incubation (Kikuzaki H, Nakatani N. 1993. Antioxidant effects of some ginger
constituents. J Food Sci 5%: 1407-1410). The extent of inhibiting the oxidation of lipids by the
herbal compounds was determined as percent inhibition. The percent inhibition of lipid
peroxidation activity by various herbals is given in Table 3. It is quite evident that the aqueous
extract of Ziziphus mauritiana is superior in LPO inhibition than any other herbal extract studied
in this project.

Table 3: Percent inhibition of LPO by herbal extracts

Note: AQ -Aqueous extract, PE- Petroleum Ether extract,
ET- Ethanol extract, ME- Methanol extract
Example 3
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.
In the standard protocol, the 0.1 mM DPPH was prepared in methanol. The plant extracts were
added to DPPH, the mixture was incubated at 25°C for 5 mins. The absorbance was read at 520
nm (Gyamfi MA, Yonamine M, Aniya Y.1999, Free radical scavenging action of medicinal
herbs from Ghana Thonningia Sanguinea on experimentally induced liver injuries. Gen
Pharmacol 32: 661-667). Lower the absorbance, higher the antioxidant potential. The percentage
inhibition of the radical scavenging activity was calculated from the absorbance of corresponding
control and test values. The results are depicted in Fig 2 D.
Example 4
Plants reserve their excess energy in the form secondary metabolites to protect themselves from
the adverse biotic and abiotic stresses. Phenols are one such class of secondary metabolites in
turn classified as many groups like flavonoids, isoflavones, anthraquinones, coumarins and
catechins. In literature it has been stated that these molecules are the potent antioxidants
(Ivanova D, Gerova D, Chervenkov T et al, 2005. Polyphenols and antioxidant capacity of
Bulgarian medicinal plants. J. Ethnopharmacol 95: 145-150). In such aspect, the quantification of
total phenols in the aqueous and ethanolic extracts of Ziziphus mauritiana will support the lipid
peroxidation inhibition assay and DPPH radical scavenging assay results.
The total phenol content was carried out by following the folins method on both the aqueous and
ethanol extracts of the Ziziphus mauritiana leaves. The results are as follow:
Table 4: Total phenolic content in Ziziphus mauritiana leaf extracts.


The above results show that the total phenol content in the aqueous extract is 36 mg/g of the leaf
and that in the ethanol extract is 14 mg/g of the leaf. This finding is in good agreement with the
fact that the anti-oxidant activity is much higher in the aqueous extract when compared to the
ethanol extract; (higher the phenol concentration higher is the antioxidant activity).
Detailed Description of the Accompanying figures
FIGURE 1A. The percent inhibition of tyrosinase enzyme activity by various herbal extracts is
given in Figure 1A. It is evident from these data that Ziziphus mauritiana alcohol extract is
superior than any other herbal species employed in the current study in its tyrosinase inhibition
property. It is noteworthy to mention that Ziziphus mauritiana indeed is better than a
commercially used skin whitening licorice extract in this respect.
FIGURE 1B. The percent inhibition of tyrosinase enzyme activity by the bark, leaf, and fruit
extracts of Ziziphus mauritiana are given in Figure 1B. It is evident from these data that Ziziphus
mauritiana leaf extract in alcohol is superior than either bark or fruit extract.
FIGURE 1C. The percent inhibition of tyrosinase enzyme activity by different solvent extracts
the Ziziphus mauritiana leaves namely aqueous, ethanol and petroleum ether are given on Figure
1C. It is evident from these data that the ethanolic extract of Ziziphus mauritiana leaves are
superior when compared to other extracts, extracted in different solvents.
FIGURE 1D. The percent inhibitions of tyrosinase enzyme activity in the ethanolic extract of
Ziziphus mauritiana leaves are concentration dependent. Its shown in the Figure 1D. From these
data, the concentration of plant material required to exert maximum inhibition can be inferred.
FIGURE 2A. The percent inhibition of lipid peroxidation activity by various herbal extracts is
given in Figure 2A. It is quite evident that the Ziziphus mauritiana aqueous extract is again
superior in LPO inhibition than any other herbal extract studied in this project.
FIGURE 2B. This graph describes the LPO inhibition activity of the extracts of leaf, bark and fruit
of Ziziphus mauritiana in all the solvents namely aquesous, ethanol and petroleum ether. In
particular, Ziziphus mauritiana leaves showed better activity than the bark or the fruit. The
values in the graph shows the maximum anti-oxidant activity observed of these solvents.

FIGURE 2C. The percent inhibition of lipid peroxidation activity by different solvent extracts
the Ziziphus mauritiana leaves namely aqueous, ethanol and petroleum ether are given on Figure
2 C. It is evident from these data that the aqueous extract of Ziziphus mauritiana leaves are
superior when compared to other extracts extracted in different solvents.
FIGURE 2D. The percent inhibition of DPPH radical scavenging activity by the leaf extract of
Ziziphus mauritiana is indeed concentration dependent. It is shown in the Figure 2D. From these
data, the concentration of plant material required to exert maximum inhibition can be inferred.

We Claim
1. A herbal composition comprising extract of Ziziphus mauritiana selected from leaves,
bark and fruits in solvents selected from water, alcohol and petroleum ether.
2. The composition as claimed in claim 1 wherein the extract is present in amount of 0.5 to
10.0%.
3. The composition as claimed in claim 2 wherein the extract is present in amount of 0.5 to
5.0%.
4. The composition as claimed in any one of preceding claim which comprises
conventional additives selected from polyoxyethylene (2) strearyl ether, polyethylene
(21) stearyl ether and isopropyl palmitate, staeric acid, cetostearyl alcohol, mineral oil
and propylene glycol.
5. The composition as claimed in claim 1 comprising ethanol extract of Ziziphus
mauritiana adapted to provide tyrosinase inhibition.
6. The composition as claimed in claim 1 comprising aqueous extract of Ziziphus
mauritiana adapted to provide anti-oxidant effects.
7. A process for preparation of a herbal composition comprising extract of Ziziphus
mauritiana leaves/bark/fruits comprising steps of hot mixing, emulsification followed
by cooling.
8. The process as claimed in claim 7 further comprising the steps of heating the powdered
solid of the herb with the solvent, followed by filtration, cooling and centrifugation.
9. A herbal composition as herein described with reference to the accompanying examples
and figures.
10. A process for preparation of herbal composition as herein described with reference to
accompanying examples and figures.

The invention provides a herbal formulation related to skin care products useful for addressing
the skin beneficial properties exhibited by Ziziphus mauritiana extracts, like skin lightening, and
anti-aging which are evaluated by several standard in-vitro and biochemical assays. It is
ascertained that the leaf or bark or fruit extracts of Ziziphus mauritiana contain compounds
capable of blocking/suppressing one or more steps in the biochemical pathways of melanin
synthesis. In the present work, it was observed that the percentage inhibition of tyrosinase by
Ziziphus mauritiana alcohol extract was much higher than that exhibited by licorice extracts.
The present invention is directed towards the application of various herbal extracts, particularly
of Ziziphus mauritiana leaves to inhibit the tyrosinase enzyme, to protect the skin from aging
through the anti-oxidant (studied by linoleic acid as a lipid bilayer model system and DPPH as a
radical model system) property.