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Evaluation of skin corrosive property of ethanolic leaf extract of Acyranthus

aspera by in vitro Transcutaneous Electrical Resistance Test and Human skin
Model Test.

Article  in  Journal of Pharmacy Research · January 2014

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 Kuntrapakam Hema Kumar et al. / Journal of Pharmacy Research 2014,8(2),113-117
Research Article Available online through
ISSN: 0974-6943 www.jpronline.info
Evaluation of skin corrosive property of ethanolic leaf extract of Acyranthus aspera
by in vitro Transcutaneous Electrical Resistance Test and Human skin Model Test.
Kuntrapakam Hema Kumar1, 2, Dr. Gajanan Rajpal Deshmukh1, Poojari Venkata Suresh Reddy1, 2,
Boddapati Srinivasa Rao1, Suryadevara Surya Narayana1, Chirumamilla Venkata Satish Kumar1
and Dr. Lokanatha Valluru†*.
1
Scientist, Toxicology, Aptus Biosciences Private Limited, Mahabubnagar – 509002, India.
2
Research scholar, Department of Biotechnology, Dravidian University, Kuppam – 517425, India.
†
Assistant professor, Department of Biotechnology, Dravidian University, Kuppam – 517425, India.

Received on:26-11-2013; Revised on: 19-12-2013; Accepted on:12-01-2014

ABSTRACT
Objective: The present article is an attempt to investigate the corrosive property of ethanolic leaf extract of Achyranthus aspera (Amaranthaceae)
by in vitro transcutaneous electrical resistance (TER) test and in vitro human skin model test before conducting wound healing experiments
on rats to know instead of wound healing activity, whether it is having skin corrosion property or not. Methods: In TER test, application of
extract for up to 24 hours to the epidermal surfaces of skin discs in a two-compartment test system in which the skin discs function as the
separation between the compartments. Corrosive substances will be identified by their ability to produce a loss of normal stratum corneum
integrity and barrier function, which was measured as a reduction in the TER below a threshold level (5 KΩ). In human skin model test, the
extract is applied topically to a three-dimensional human skin model, comprising reconstructed epidermis with a functional stratum corneum
for 1 hour. Corrosive substances are identified by their ability to penetrate the stratum corneum by diffusion or erosion, and cytotoxic to the
underlying cell layers to produce a decrease in cell viability below threshold levels (15 %). Results: The ethanolic extract of Achyranthus
aspera does not show corrosive property in both transcutaneous electrical resistance and human skin model test. Conclusion: The results
obtained in these studies suggest a significant scope for the isolation of biological active components for further studies.

KEY WORDS: Epidermis, EpiSkin, MTT, Stratum corneum, Transcutaneous electrical resistance.
INTRODUCTION
Skin corrosion refers to the production of irreversible tissue damage MATERIALSAND METHODS
in the skin following the application of a test substance [1, 2]. The use
of laboratory animals in the assessment of skin corrosivity is very Plant material & Preparation of leaf extract
common. This involves pain and suffering to the animals, hence, to The leaves of Achyranthes aspera were freshly collected during Janu-
determine the corrosive property of the chemicals various alterna- ary-March in and around the Yenugonda village (Mahabubnagar Dis-
tives in vitro methods were used [3-5]. An important consideration in trict, Andhra Pradesh, India) and were cleaned with distilled water
cosmetic innovation and toxicology is the growing concern about and shade dried at room temperature. The dried leaves were pow-
the ethics of testing final/finished products on animals, and it is gradu-
dered (100 g) and were extracted separately to exhaustion in a soxhlet
ally being discouraged and alternative methods are being designed apparatus using ethanol as a solvent system. The extract was filtered
[6]
. through Whatman filter paper No.1 and then concentrated by evapo-
rating at the low temperature (40-50 °C) to get a 2.59 g yield. The
Achyranthes aspera is an important medicinal herb known as a weed extract was preserved in an airtight container at 4±2 °C until further
throughout India. However, in traditional systems of medicines al- use.
most all parts of it are used. Seeds, roots and shoots are the most
important parts, which are used by traditional healers for the treat- Preliminary qualitative analysis of phytochemicals in leaf extract
ment of fever, dysentery and diabetes [7-9]. The present article is an A preliminary qualitative phytochemical analysis of leaf extract of
attempt to evaluate the corrosive property of ethanolic leaf extract of Achyranthes aspera was carried out [10].
Achyranthus aspera by in vitro skin corrosion assay using rat skin
and reconstituted human epidermis skin model. Sodium hydroxide Test:
*Corresponding author. Small quantity of the extract was treated with sodium hydroxide solu-
Dr. Lokanatha Valluru tion; Formation of yellow color indicates the presence of Flavonoids.
Department of Biotechnology, School
Foam Test:
of Herbal Studies & Naturo Sciences,
The extract was diluted with 20 ml of distilled water and agitated in the
Dravidian University, graduated cylinder for 15 minutes. A one-centimeter layer of foam
Kuppam – 517 426, indicates the presence of saponins.
Andhra Pradesh, India.
Journal of Pharmacy Research Vol.8 Issue 2.February 2014 113-117
 Kuntrapakam Hema Kumar et al. / Journal of Pharmacy Research 2014,8(2),113-117
Haemolysis Test: skin disc was tied to hold in place with suturing thread. Excess tissue
About 2 ml of blood was taken in two test tubes separately. In one was trimmed and sealed with petroleum jelly.
test tube equal quantity of water and in another test tube, an equal
quantity of extract dissolved in water was added. Presence of clear (b) Human Skin Units
red liquid in the first tube, indicates haemolysis, if same is the case EpiSkin, the human skin units were obtained from EpiSkin SNC,
with second test tube (with extract) indicates the presence of sapo- France, which was used in this study. Lot number 10-EKIN-028.
nin.
EpiSkin is a three-dimensional reconstituted human epidermis model
Mayer’s Test: comprising the main basal, supra basal, spinosus and granular layers
To a few ml of extract, two drops of Mayer’s reagent was added by the and a functional stratum corneum.
sides of the test tube. A white or creamy precipitate indicates the
presence of alkaloids. EXPERIMENTAL PROCEDURES
Wagner’s Test: 1.Transcutaneous Electrical Resistance Test
To a few ml of extract, few drops of Wagner’s reagent (Iodine 1.278 g In this test, the transcutaneous electrical resistance was measured
and potassium iodide 2 g dissolved in 100 ml of distilled water) was with Millicell®ERS-2, Epithelial Volt-Ohm meter, MILLIPORE, USA.
added by the side of the test tube. The presence of a reddish brown Prior to the test, the electrical resistance of two prepared skin discs
precipitate indicates the presence of alkaloids. was measured as a quality control procedure for each animal skin.
Skin discs with resistance values greater than 10 kΩ (10000Ω) were
Hager’s Test: only used for the study.
To a few ml of extract, 2 ml of Hager’s reagent (Saturated aqueous
solution of picric acid) was added. A prominent yellow precipitate Three skin discs per group were used. In group 1 (G1), the skin disks
indicates the presence of alkaloids. were treated with distilled water as a negative control. The skin discs
from group 2 and 3 (G2 & G3) treated with 10M hydrochloric acid and
Molish’s Test: acrylic acid respectively as positive control. While, the skin discs
To two ml of extract, Two drops of the alcoholic solution of a-naph- from group 4 (G4) were treated with extract. The controls and extract
thol was added, the mixture was shaken well and 1 ml of concentrated were applied to the epidermal surface of the skin disc at 20 – 23 0C for
sulphuric acid is added slowly along the sides of the test tube and 24 hours and then washed with distilled water. TER was measured by
allowed to stand. A violet ring indicates the presence of carbohy- pouring a 154mM solution of MgSO4 into the tube and lower reser-
drates. voir (surrounding the tube). The data bridge electrodes were placed
on either side of the skin disc to measure the resistance. The inner
Ferric Chloride Test: electrode was placed inside the tube during resistance measurement
Two grams of extract were boiled with 5 ml of 45 % ethanol for 5 to ensure that a constant length of the electrode was submerged in
minutes. The mixture was cooled and filtered. 1 ml of filtrate was the MgSO4 solution. The outer electrode was positioned inside the
diluted with distilled water and added 2 drops ferric chloride. A tran- receptor chamber so that it rests on the bottom of the chamber. The
sient greenish to black color indicates the presence of tannins. TER values of negative control, positive control and treated skin
discs were recorded.
Test System
The transcutaneous electrical resistance test and human skin model 2. Human Skin Model Test
test were performed by using (a) rat skin and (b) EpiSkin (reconsti- In this test, after receiving of skin units from the EpiSkin SNC, France,
tuted human epidermis) respectively. quality check was performed to see the suitability of the skin units for
the study. When found suitable the skin units transferred to fresh
(a) Rat skin disc preparation maintenance medium and kept in incubation at 37 0C, 5 % CO2 and 95
Transcutaneous electrical resistance test using rat skin has been % humidity for 24 hours.
approved by the IAEC on 10th February 2010.
Liquid test items 12 µl directly or 12 ± 2 mg of solid materials with 5 µl
Normal, young and healthy inbred female Wistar rats of 20±2-day-old of distilled water were applied to the epidermal surface in order to
were obtained from Central animal house, Aptus Biosciences Private improve further contact with gentle spreading to cover all surface
Limited (CPCSEA Registration Number 1312/c/09/CPCSEA). Hairs from areas.
the dorsal and flank region of female rats were removed carefully with
small clippers. The animals have given a wash with antibiotic solu- Three skin units per group were used. Group 1 (G1) the skin units was
tion twice with an interval of three days to inhibit bacterial growth, treated with sodium chloride 0.9 % as negative control. However, the
before initiation of the experiment [2]. Animals were humanely sacri- skin units from group 2 and 3 treated with 8 N potassium hydroxide
ficed and the dorso-lateral skin of each animal was removed. The and acrylic acid respectively as positive controls and group 4 (G4)
excess subcutaneous fat stripped off carefully by peeling it away skin units were treated with extract. The epidermis were allowed to be
from the skin. Circular skin discs, with a diameter of approximately 20 in contact with the test items for 60 minutes at room temperature (19
mm each was removed from the skin. Each skin disc was placed over to 28 0C) and then rinsed with 25 ml phosphate buffered saline. After
one of the ends of a tube (internal diameter of the tube approx. 10 mm) washing, the skin units were transferred to fresh maintenance medium
ensuring that the epidermal surface was in contact with the tube. The under sterile condition and incubated at 37 0C, 5 % CO2 and 95 %

Journal of Pharmacy Research Vol.8 Issue 2.February 2014 113-117

 Kuntrapakam Hema Kumar et al. / Journal of Pharmacy Research 2014,8(2),113-117
humidity for 42 hours. After incubation, the skin units were transferred zation of the epidermal squamous cells. All the treated groups were
to the next wells of same 12 well plate with 2 ml of MTT solution (0.3 assessed blindly by the pathologist and the results were compared
mg / ml) in the assay medium. The plate was incubated for 3 hours at with the negative control and positive control groups.
5 % CO2 and 95 % humidity. After the final incubation, the skin units
were removed and punched biopsy was carried out, the biopsy material RESULTS
was transferred in prelabeled tubes containing 500 µl of acidic Preliminary qualitative phytochemical analysis of ethanolic extract of
isopropanol. These tubes were kept overnight for formazan extraction Achyranthus aspera revealed positive for Flavonoids, Saponins,
at 4 0C. After extraction, the tubes were centrifuged at 500 RPM for 10 Alkaloids, Carbohydrates (Table I).
minutes. 200 µl solution from each tube was transferred to two different
wells in 96 well flat-bottom microtiter plate as aliquot 1 and aliquot 2. Transcutaneous electrical resistance was measured for Achyranthus
The optical density of each sample was measured by 570 nm using a aspera extract, Positive controls and negative control. Mean TER
multimode microplate reader (SYNERGY - 4). The percent cell viability values of negative control (distilled water) and extract treated skin
of each skin unit was calculated by a formula discs were observed more than 10 kΩ i.e. 12235.67 and 11461 with no
obvious damage and hence considered non-corrosive. However, TER
Mean OD of respective skin units value of positive control 10 M hydrochloric acid & acrylic acid treated
Percent cell viability = —————————————— X 100 skin discs were observed less than 5 kΩ i.e 327.33 and 355 with
Mean OD of negative control obvious damage, hence considered as corrosive (Table II).

BASIC HISTOLOGY In the human skin model test, the OD values of negative control,
Skin specimens used in the transcutaneous electrical resistance were positive controls and extract were recorded and the percent cell vi-
removed from the tubes after measuring TER for histological exami- ability was calculated. The percent cell viability of negative control
nation. The tissues were processed routinely for light microscopy and extract treated skin units were observed more than 50 percent i.e
(fixation in 10% buffered formaldehyde, dehydration, paraffin embed- 100.00 and 94.71 at 60 minutes of exposure time, hence considered
ding, sectioning [5 micrometer], and staining). Hematoxylin-eosin was non-corrosive. However, percent cell viability of positive control (8 N
used for basic staining. The tissue samples were evaluated for the potassium hydroxide and Acrylic acid) was less than 15 percent i.e
following histological criteria; Integrity of stratum corneum, organi- 3.81 and 2.97. Hence, considered as corrosive (Table III).
Table I: Results of preliminary phytochemical analysis of Ethanolic extract of Achyranthus aspera.
Test Observation Inference

Sodium Hydroxide Test Formation of yellow color Presence of flavonoids

Foam Test Formation of 1 cm of foam Presence of saponins
Haemolysis Test Clear red liquid indicates haemolysis Presence of saponins
Mayer’s Test No white or creamy precipitate Presence of alkaloids
Wagner’s Test No reddish brown precipitate Presence of alkaloids
Hager’s Test Formation of yellow precipitate Presence of alkaloids
Molish’s Test Formation of violet ring Presence of carbohydrates
Ferric Chloride Test No transient greenish to black color Absence of tannins
Table II: Transcutaneous electrical resistance values of skin Table III : Mean percent cell viability of skin units of each group.
disc in each group.
Group S k i n Optical Density Blank corrected OD Mean of % Cell Viability
Group Skin Electrical Mean± SD Visual
Unit Aliquot Aliquot Aliquot Aliquot corrected Relative Mean
disc No. Resistance (Ω) damage No. 1 2 1 2 OD

G1 1 11425 12235.67 ± 1185.28 No Blank 1 0.033 0.032 0 0 0.032 - -

2 13596 2 0.033 0.033 0 0 0.033 -
3 11686 3 0.032 0.032 0 0 0.032 -
G2 1 350 327.33 ** ± 32.57 Yes G1 1 0.748 0.534 0.716 0.502 0.609 98.54 100.00
2 342 2 0.714 0.575 0.682 0.543 0.612 99.11
3 290 3 0.654 0.675 0.622 0.643 0.632 102.35
G2 1 0.055 0.06 0.023 0.028 0.025 4.05 3.81 **
G3 1 325 355** ± 56.34 Yes
2 0.054 0.054 0.022 0.022 0.022 3.48
2 420 3 0.056 0.057 0.024 0.025 0.024 3.89
3 320 G3 1 0.048 0.049 0.015 0.017 0.016 2.59 2.97 **
G4 1 11564 11461ns ± 125.65 No 2 0.058 0.052 0.025 0.02 0.022 3.64
2 11321 3 0.047 0.051 0.014 0.019 0.016 2.67
3 11498 G4 1 0.612 0.662 0.547 0.587 0.567 91.82 94.71 *
2 0.587 0.562 0.625 0.598 0.612 99.03
3 0.551 0.565 0.551 0.601 0.576 93.28
# The values in the table are rounded off to the nearest digit # The values in the table are rounded off to the nearest digit value.Blank (Acidic
value.G1- Distilled Water (Negative Control); G2 – 10 M HCl isopropanol); G1 - Negative control (0.9 % NaCl); G2 - Positive control (8 N KOH);
(Positive Control-1); G3 - Acrylic acid (Positive Control-2); G4 G3 - Positive control (Acrylic Acid); G4 - Extract treated.One-way ANOVA with
- Extract treated. One-way ANOVA with Dunnett’s post-test was Dunnett’s post-test was performed P< 0.05 was considered statistically significant.
performed P< 0.05 was considered statistically significant. **
= P < 0.01, ns= P > 0.05.
**
= P < 0.01, ns= P > 0.05.
Journal of Pharmacy Research Vol.8 Issue 2.February 2014 113-117
 Kuntrapakam Hema Kumar et al. / Journal of Pharmacy Research 2014,8(2),113-117

Histopathological findings Figure II: Skin disc treated with 10 M Hydrochloric acid
Histopathology was performed to skin discs used in the transcutane-
ous electrical resistance test. All the groups were subjected to histo-
pathology, No histopathological findings were observed in negative
control and extract treated skin discs (fig I), whereas destruction of
stratum corneum, stratum granulosum, stratum spinosum and stra-
tum basale was observed both the group skin discs treated with
positive controls these are the four layers present in the epidermis.
Stratum basale is the bottom layer of epidermis these cells are a kind
of stem cells, which generates proliferating keratinocytes that mi-
grates towards stratum corneum in the process of cell shedding. Dur-
ing this process these cells undergo some modifications like accumu-
lation in the stratum spinosum, enucleated and undergo death in
stratum granulosum and finally reaches stratum corneum and acts as
a barrier to the internal tissues. From the microphotographs (fig II &
III) treated with positive controls we can clearly see the destruction
and necrosis of these cells in the stratum basale and stratum spinosum,
which created irreversible damage, is corrosive to skin.

STATISTICAL ANALYSIS Legend : Figure II treated with 10M HCl showing the erosion
Statistical analysis was performed by using GraphPad InStat version stratum corneum and the destruction of Stratum granulosum,
3.00 for Windows 95, GraphPad Software, San Diego California USA, Stratum spinosum and Stratum basale which is clear indication of
www.graphpad.com. The electrical resistance in transcutaneous elec- irreversible damage to the skin.
trical resistance test and % cell viability in human skin model test was
compared to the results of the negative control group by one-way Figure III: Skin disc treated with Acrylic acid
ANOVA with Dunnett’s post-test was applied to determine the statis-
tical significance of the results, and a value of P < 0.05 was consid-
ered significant.

Figure I: Skin disc treated with ethanolic extract of Achyranthus

aspera

Legend : Figure III treated with Acrylic acid showing the erosion
stratum corneum and the destruction of Stratum granulosum,
Stratum spinosum and Stratum basale which is clear indication of
irreversible damage to the skin.

CONCLUSION
From the present study, the in vitro skin corrosion assays by trans-
cutaneous electrical resistance test; ethanolic leaf extracts of
Achyranthus aspera showed TER values greater than 10 kΩ with no
Legend : Figure I treated with ethanolic extract of Achyranthus
visual damage to the skin. Similarly, in human skin model test, the
aspera showing the normalstratum corneum, Stratum granulosum,
mean percent cell viability of the extract treated skin units was greater
Stratum spinosum and Stratum basale which clearly indicate the
extract is non-corrosive to the skin. than 50 %. Hence, it can be concluded that the ethanolic leaf extract

Journal of Pharmacy Research Vol.8 Issue 2.February 2014 113-117

 Kuntrapakam Hema Kumar et al. / Journal of Pharmacy Research 2014,8(2),113-117
of Achyranthus aspera has no skin corrosive property, which could 5. Christina Grindon, Robert Combes, Mark T.D. Cronin, David
be beneficial in different skin preparations and are a subject for future W. Roberts and John F.Garrod. ATLA 35, 673–682, 2007.
studies. 6. Nigam PK. Adverse reactions to cosmetics and methods of
testing. Indian J Dermatol Venereol Leprol. 2009;75:10-19.
ACKNOWLEDGEMENTS 7. Saurabh Srivastav, Pradeep Singh, Garima Mishra, K. K. Jha,
The authors thank to Dr. Praveen Bommu and Dr. T. Koti Reddy,
R. L. Khosa, Achyranthus aspera – An important medicinal
Aptus Biosciences Private Limited, SVS medical college campus,
Yenugonda, Mahabubnagar for providing facilities to carry out these plant: A review, J. Nat. Prod. Plant Resour., 2011, 1 (1): 1-14
experiments. 8. Girach RD, Khan ASA . Ethnomedicinal uses of Achyranthes
aspera leaves in Orissa (India). Int J Pharmacogn.1992, 30 :
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Source of support: Nil, Conflict of interest: None Declared

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