FIELD OF THE INVENTION
The present invention relates to the preparation of lipid substrate (s) and a method
for determining the mildness of surfactants.
BACKGROUND OF INVENTION
The skin is the largest organ of the body, protects us from microbes and the
elements, helps in regulating body temperature and permits the sensation of touch,
heat and cold. The skin has three layers; epidermis (outermost layer of skin) which
provides a waterproof barrier and creates our skin tone, dermis (beneath the
epidermis) that contains tough connective tissue, hair follicles and sweat glands,
and the subcutaneous tissue (hypodermis) which is made of fat and connective
tissue.
The epidermal layer of skin is comprised of five layers; Stratum Corneum which
is composed of 10 to 30 layers of flattened dead corneocytes (final step of
keratinocyte differentiation), a translucent layer (Stratum Lucidum) in palms and
soles, granular layer (Stratum Granulosum), spinous layer (Stratum Spinosum)
and basal/germinal layer (Stratum Basale).
The stratum corneum, located on the outermost layer of skin, protects the body
from contact and infiltration of foreign matter such as bacteria and hazardous
substances. Reduction of moisture content in stratum corneum creates a dry,
roughened skin condition. The intercellular lipids in the stratum corneum are
composed mainly of ceramides, free fatty acids and cholesterol, and are known to
form a lamellar structure.
Surfactants are compounds that lower the surface tension between two liquids or
between a liquid and a solid. Surfactants may act as detergents, wetting agents,
emulsifiers, foaming agents, and dispersants. Surfactants have a hydrophobic part
and a hydrophilic part. The hydrophobic part consists of an uncharged
carbohydrate group that can be straight, branched, cyclic or aromatic. Depending
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on the nature of the hydrophilic part the surfactants are classified as anionic, nonionic,
cationic or amphoteric. Surfactants penetrate the stratum corneum and are
absorbed into the keratin of cornified cells, mixing with intercellular lipids.
Surfactants are also known to remove cutaneous lipids such as fatty acids, fatty
acid glycerides and cholesteryl esters, thus causing damage to the skin even if the
removed lipids are minimal. Interaction of surfactants with the stratum corneum
proteins leads to macroscopic swelling of the membrane. The swelling of the
stratum corneum is more with anionic surfactants compared to that with cationic
and non-ionic surfactants. The effects of surfactants on stratum corneum
intercellular lipids have been an object of interest in the past in connection with
the skin barrier function.
In the stratum corneum, the physical conformation of the intracellular lamellar
lipids provides a tight and semi-permeable barrier to the passage of water through
the tissues. Surfactant removal of lipid components can alter the optimum level
required to maintain a healthy stratum corneum.
United States patent application No. 20130000394 discloses a substrate
mimicking intercellular lipids in stratum corneum consisting of a substrate and a
lipid membrane formed on the substrate, wherein the lipid membrane is formed
from ceramide, palmitic acid and cholesterol, and the ceramide, palmitic acid and
cholesterol are present in a mass ratio of 20-70:10-600:20-40 (ceramide : palmitic
acid : cholesterol).
Various methods have been developed for detecting lipid loss on interaction with
surfactants in vitro. According to one method, the ability of a surfactant to extract
stearic acid and cholesterol is assessed by determining the solubility of both these
lipids in a 5% w/w surfactant solution. Another method involves a liposome
solubilization assay that estimates the ability of a surfactant to alter the
permeability parameter of a lipid bilayer. Hence, such a method is a better
simulation of the surfactant skin interaction in vivo. The mildness of surfactants
herein is calculated with the help of HPLC or GC techniques. However, both the
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methods mentioned herein are inconsistent, lengthy and have low accuracy level
(statistical errors in measurement of data).
Due to the aforementioned drawbacks, there exists a need in the art for a method
to determine the mildness of surfactants that is not only more accurate and precise
compared to previous known methods but also easy to perform in a laboratory.
The present invention is directed towards a novel process for the preparation of
lipid substrates and a method to determine the mildness of surfactants by
estimating lipid loss.
SUMMARY OF THE INVENTION
In order to precisely determine the effects of surfactants on the skin, the present
inventors have developed a simple skin model, for the stratum corneum,
comprising lipids present in the intercellular spaces of the stratum corneum.
Furthermore, the inventors have also deviced a method to determine the mildness
of surfactants.
In accordance with an embodiment of the invention, there is provided a method to
determine the mildness of surfactants, comprising the steps of: (a) measuring
initial weight of a lipid substrate; (b) exposing the lipid substrate to a solution of
the surfactant in water for a pre-determined period; (c) rinsing the lipid substrate
with water; (d) drying the lipid substrate at a temperature ranging between 105
°C-110 °C; (e) measuring final weight of the lipid substrate and (f) calculating the
difference between the initial weight and the final weight of the lipid substrate.
In accordance with another embodiment of the invention, the surfactant is selected
from the group consisting of an anionic surfactant, an amphoteric surfactant, a
non-ionic surfactant, a cationic surfactant and combinations thereof.
In accordance with another embodiment of the invention, the lipid substrate used
for determining the mildness of surfactants is prepared by a process, comprising
the steps of: (a) selecting at least one lipid from the group consisting of stearic
acid, soya lecithin and cholesterol; (b) melting and mixing the lipids in a
5
predetermined ratio and adding a suitable solvent to form a mixture; and (c)
evaporating the suitable solvent from the mixture to obtain the lipid substrate.
In accordance with another embodiment of the invention, the suitable solvent is
any solvent capable of dissolving lipids.
In accordance with another embodiment of the invention, the suitable solvent is
diethyl ether or chloroform.
In accordance with another embodiment of the invention, the lipid substrate is in
the form of a tablet.
In accordance with another embodiment of the invention, there is provided a lipid
substrate, comprising: (a) stearic acid; (b) soya lecithin; and (c) cholesterol in a
mass ratio ranging from 25.33-66.66 : 13.33-46.66 : 26.66-46.66.
BRIEF DESCRIPTION OF DRAWINGS
Figure 1. illustrates the lipid substrate comprising stearic acid, soya lecithin and
cholesterol in the form of tablets.
DETAILED DESCRIPTION OF THE INVENTION
Discussed below are some representative embodiments of the present invention.
The invention in its broader aspects is not limited to the specific details and
representative methods. The illustrative examples are described in this section in
connection with the embodiments and methods provided. The invention according
to its various aspects is particularly pointed out and distinctly claimed in the
attached claims read in view of this specification.
It is to be noted that, as used in the specification and the appended claims, the
singular forms "a," "an," and "the" include plural referents unless the context
clearly dictates otherwise. Thus, for example, a reference to a composition
containing “a compound” includes a mixture of two or more compounds. It should
also be noted that the term “or” is generally employed in its sense including
“and/or” unless the content clearly dictates otherwise.
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The expression of various quantities in terms of “%” or “% w/w” means the
percentage by weight of the total solution or composition unless otherwise
specified.
All cited references are incorporated herein by reference in their entireties.
Citation of any reference is not an admission regarding any determination as to its
availability as prior art to the claimed invention.
The present invention, in its product and process aspects, is described in detail as
follows:
Surfactants are known to remove cutaneous lipids, thereby causing inflammation
and damage to the skin. Moreover, it is becoming increasingly difficult to obtain
intercellular lipid samples from animals, for the purpose of testing cosmetics, in
light of animal protection. Hence, there is a need for a novel in vitro model
substrate that not only mimics the intercellular lipids of the stratum corneum but
also enables estimation of lipid loss on exposure to surfactants. Such a novel
substrate would be very useful in evaluating various surfactants present in
personal care and cosmetic products. Accordingly, the present invention relates to
a process for preparing a lipid substrate and a method to determine the mildness of
surfactants. The lipid substrate is prepared by melting and mixing at least one lipid
selected from the group consisting of stearic acid, soya lecithin and cholesterol in
a predetermined ratio, in the presence of a suitable solvent. Stearic Acid, Soya
Lecithin, and Cholestrol were mixed in a mass ratio ranging from 25.33-66.66 :
13.33-46.66 : 26.66-46.66.
Thereafter, the warm mixture is poured into petri dishes and allowed to cool to
room temperature, to a suitable form.
The term “lipid” used herein refers to a large and diverse group of naturally
occurring organic compounds that are related by their solubility in nonpolar
organic solvents (e.g. ether, chloroform, acetone & benzene) and general
insolubility in water. Stearic Acid (IUPAC systematic name: Octadecanoic
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acid) is a saturated long-chain fatty acid with an 18-carbon backbone. It is a waxy
solid that is obtained from many animal and vegetable fats and oils.
The term “Soya lecithin” used herein refers to a composition extracted from
soybeans and consists of a mixture of three main phospholipids
(phosphatidylcholine, phosphatidylethanolamine and phosophotidylinositol),
glycolipids, neutral lipids and carbohydrates. The term “Phospholipids” used
herein refers to a class of lipids that are a major component of all cell membranes.
They can form lipid bilayers because of their amphiphilic characteristic. The
structure of the phospholipid molecule generally consists of two hydrophobic fatty
acid "tails" and a hydrophilic "head" consisting of a phosphate group. The two
components are joined together by a glycerol molecule. The phosphate groups can
be modified with simple organic molecules such as choline.
The term “Phosphatidylcholine” used herein refers to a class of phospholipids that
incorporate choline as a headgroup. They are a major component of biological
membranes and can be easily obtained from a variety of readily available sources,
such as egg yolk or soybeans, from which they are chemically extracted
using hexane. The term “phosphatidylethanolamine” used herein refers to a class
of glycerophospholipids in which a phosphatidyl group is esterified to the hydroxy
group of ethanolamine. The term “phosophotidylinositol” used herein refers to a
class of glycerophospholipids in which a phosphatidyl group is esterified to one of
the hydroxy groups of inositol.
“Cholesterol”, IUPAC name: (3β)-cholest-5-en-3-ol, refers to
a sterol (or modified steroid) and a lipid molecule, which is biosynthesized by all
animal cells because it is an essential structural component of all animal (not plant
or bacterial) cell membranes that is required to maintain both membrane structural
integrity and fluidity.
The term “suitable solvent” used herein refers to any solvent capable of dissolving
lipids, such as chloroform, ethanol, diethyl ether and so forth, each of which can
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be used individually or in combination. The suitable form of the lipid substrate is
a tablet.
The present invention is more particularly described in the following non-limiting
examples that are intended as illustrations only since numerous modifications and
variations within the scope of the present invention will be apparent to a skilled
artisan. Unless otherwise noted, all parts, percentages, and ratios reported in the
following examples are on a weight basis, and all reagents used in the examples
were obtained or made available from the chemical suppliers.
Examples 1-5
Table 1 lists the constituents of the lipid tablet and their amounts. The unit of each
value is weight percent.
Table 1. Composition of the lipid tablet
Constituents
Example
1
% w/w
Example
2
% w/w
Example
3
% w/w
Example
4
% w/w
Example
5
% w/w
Stearic Acid 27.3 38.5 46.7 52.9 55.6
Soya Lecithin 27.3 23.1 20.0 17.6 16.7
Cholesterol 45.5 38.5 33.3 29.4 27.8
Example 6
Process for preparing a lipid tablet
A lipid substrate in the form of a tablet was prepared by a process given herein
below. Stearic acid, soya lecithin and cholesterol were weighed and mixed in
amounts provided in Table 1. Diethyl ether was added to the mixture and mixing
was continued over a water bath to form a uniform mixture. The warm lipid
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mixture was poured into tablet molds and allowed to cool to room temperature.
After standing for another 30 minutes, the tablets were stripped out of their molds.
The lipid tablets were uniform, durable and insoluble in water. The lipid tablets of
Example 3 were used for evaluating the mildness of surfactants in Example 7.
Figure 1 illustrates the lipid tablets of Example 3 made by the above process.
The mildness of surfactants can be determined by calculating the loss of lipids
from the lipid substrate, on exposure to a surfactant solution.
The loss of lipids from the lipid tablet is an indicator of the mildness of the
surfactant solution. The term “mildness” used herein refers to the ability of the
surfactant solution to dissolve lipids from a lipid substrate. A surfactant solution
which is milder dissolves relatively less lipids compared to one that is less mild.
The term “surfactant solution” used herein refers to a solution of surfactant (s) in
water or to a solution in water of any personal care product such as soaps, body
wash, shampoos, shaving cream and so forth that contains a plurality of
surfactants. The term “surfactant” used herein refers to an organic compound that
is amphiphilic, meaning it contains both hydrophobic group (their tail)
and hydrophilic group (their head). Therefore, a surfactant contains both a waterinsoluble
(or oil-soluble) component and a water-soluble component.
In another embodiment of the invention, the surfactant (s) is selected from the
group consisting of an anionic surfactant, amphoteric surfactant, cationic
surfactant and a non-ionic surfactant, each of which can be used either
individually or in combination. The term “anionic surfactant” used herein refers to
a surfactant that contains an anionic functional group at its head, such
as sulfate, sulfonate, phosphate, and carboxylate. The term “non-ionic surfactant”
used herein refers to a surfactant that does not bear an electrical charge. The term
“cationic surfactant” used herein refers to a surfactant having a cationic functional
group as its head. The term “amphoteric surfactant” as used herein refers to a
surfactant that carries both a negative and positive charge.
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Example 7
A method to determine the mildness of surfactants
The percent loss of lipids from the tablet was calculated by a method described
herein below. The initial weight of a lipid tablet was recorded. A conical flask,
with a 5% surfactant solution and a lipid tablet, was placed on an orbital shaker
for 1 hour at 100 revolutions per minute. Thereafter, the lipid tablet was removed
from the surfactant solution and rinsed in de-mineralized water, with stirring using
a magnetic stirrer, for 3 minutes. The rinsed tablet was dried in a Petri dish, in a
hot air oven at 105 °C, for 2 hours. The final weight of the dried lipid tablet was
recorded.
Calculation
The percent weight of lipids lost or solubilized from the tablet was calculated as
follows:
Lipid Loss = Initial weight of lipid tablet – Final weight of the dried lipid tablet
Percent Lipid Loss = Lipid Loss/Initial weight of the tablet ×100
In order to validate the aforementioned method, the loss of lipids from the tablet
on exposure to surfactant solutions was determined. The surfactant solutions were
prepared by dissolving the following personal care products in water.
1. A shampoo containing Sodium Laureth Sulfate, TEAdodecylbenzenesulfonate,
Cocamidopropyl Betaine;
2. A body wash and shampoo containing Lauryl Glucoside,
Cocamidopropyl Betaine, Decyl Glucoside, Sodium Lauryl Glucose
Carboxylate, Glyceryl Oleate, Coco-glucoside;
3. A baby milk bath containing Sodium Laureth Sulfate, Cocamidopropyl
Betaine, Disodium Cocoamphodiacetate, Sodium Cocoamphoacetate,
Sodium Lauryl Sulphate.
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The lipid loss analysis for each of the above products was performed in triplicate
and presented in Table 2.
Table 2: Percent lipid loss on exposing lipid substrate to surfactant solution
No Product
Percentage lipid loss
Average
Standard
n1 n2 n3 deviation
1
A shampoo containing
Sodium Laureth Sulfate,
TEAdodecylbenzenesulfonate,
Cocamidopropyl Betaine
6.05 6.02 6.40 6.16 0.21
2
A body wash and
shampoo containing
Lauryl Glucoside,
Cocamidopropyl Betaine,
Decyl Glucoside, Sodium
Lauryl Glucose
Carboxylate, Glyceryl
Oleate, Coco-glucoside
3.54 2.91 3.79 3.41 0.45
3
A baby milk bath
containing Sodium
Laureth Sulfate,
Cocamidopropyl Betaine,
Disodium
Cocoamphodiacetate,
Sodium
Cocoamphoacetate,
Sodium Lauryl Sulphate
5.80 4.51 5.25 5.19 0.64
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From the results for percent lipid loss in the above table, it is evident that the
method developed herein is reproducible and confirms the validity of the method
to estimate the mildness of surfactant systems. Such a method along with the lipid
tablet prepared herein may be utilized to determine the mildness of surfactants
present in personal care and cosmetic products.
While particular embodiments of the lipid substrate of present invention have
been illustrated and described, it would be obvious to those skilled in the art that
various other changes and modifications can be made without departing from the
spirit and scope of the invention. It is thereof intended to cover in the appended
claims such changes and modifications that are within the scope of the invention.

We Claim:
1. A method to determine the mildness of a surfactant, comprising the
steps of: (a) measuring initial weight of a lipid substrate; (b) exposing
the lipid substrate to a solution of the surfactant in water for a predetermined
period; (c) rinsing the lipid substrate with water; (d) drying
the lipid substrate at a temperature ranging between 105 °C-110 °C; (e)
measuring final weight of the lipid substrate; and (f) calculating the
difference between the initial weight and the final weight of the lipid
substrate.
2. The method to determine the mildness of a surfactant as claimed in claim
1, wherein the surfactant is selected from the group consisting of an
anionic surfactant, an amphoteric surfactant, a non-ionic surfactant, a
cationic surfactant and combinations thereof, and personal care products.
3. The method to determine the mildness of a surfactant as claimed in claim
2, wherein the lipid substrate is prepared by a process, comprising the
steps of: (a) selecting at least one lipid from the group consisting of
stearic acid, soya lecithin and cholesterol; (b) melting and mixing the
lipid (s) in a predetermined ratio and adding a suitable solvent to form a
mixture; and (c) evaporating the suitable solvent to obtain the lipid
substrate.
4. The method to determine the mildness of a surfactant as claimed in claim
3, wherein stearic acid, soya lecithin and cholesterol are mixed in a mass
ratio ranging from 25.33-66.66:13.33-46.66:26.66-46.66.
5. The method to determine the mildness of a surfactant as claimed in claim
3, wherein the suitable solvent is any solvent in which the lipids are
soluble.
6. The method to determine the mildness of a surfactant as claimed in claim
4, wherein the suitable solvent is either diethyl ether or chloroform.
7. The method to determine the mildness of a surfactant as claimed in claim
6, wherein the lipid substrate is in the form of a tablet.
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8. The lipid substrate prepared by the process as claimed in claims 3 to 7.