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Original Article

Aloe vera is non-toxic to cells: A microculture

tetrazolium technique colorimetric assay study
Devi Gopakumar, Sunil Sukumaran Nair1
Department of Oral Medicine and Radiology, Noorul Islam Dental College, Trivandrum, 1Department of Oral and
Maxillofacial Pathology, Pushpagiri College of Dental Sciences, Tiruvalla, Kerala, India

ABSTRACT

Introduction: Aloe vera (Av), a succulent of Liliaceae family is now a widely used medicinal plant. Its’ application
covers a wide spectrum of human diseases, including oral mucosa, gastric mucosa and skin. Aloe vera preparations
in the form of gel, mouth washes and cream are applied topically for many oral diseases. The applications include
oral lichen planus, candidiasis, oral submucous fibrosis, geographic tongue, etc. Aims and Objectives: To evaluate
the cytotoxicity of Av on human fibroblasts. Materials and Methods: Aloe vera preparation (70%) was applied on the
fibroblast cell lineage and the cell viability was evaluated by microculture tetrazolium technique (MTT) colorimetric
assay. Results: The cell viability at different concentrations was measured. The cells have maintained their viability
at different concentrations used in the study. Conclusion: Our study shows the cell viability at different sample
concentrations of Av. This could open up wide clinical applications of Av for reactive, inflammatory and potentially
malignant oral and other mucocutaneous diseases.
Key words: Aloe vera, colorimetric assay, cytotoxicity, microculture tetrazolium technique (MTT) assay

been used for an array of ailments since ancient times

Introduction as a medicinal plant. There are more than 360 different

T
homas Alva Edison once said, “The doctor of the
future will give no medicine, but will instruct his
patient in the care of the human frame, in diet
and in the cause and prevention of disease.” The name
Aloe vera (Av) or true aloe meaning a ‘shining bitter
substance’, is perhaps derived from Arabic ‘Alloeh’,
Syrian ‘Alwai’ or Hebrew ‘Halal’.[1] The first reference
to Av in English was made in AD 1655 in a translation
from Dioscorides by John Goodyear.[1] Aloe vera is a
succulent of Liliaceae family. Aloe vera is widely used
for both commercial and therapeutic purposes. It has
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DOI:
10.4103/0972-1363.155628
species of Av. But there are about 15 poisonous species
containing a deadly hemlock-like substance; a method
chosen by Socrates to commit suicide.[2]
Aloe vera products contain constituents that accelerate
wound healing. It helps to reduce inflammation, pain
and itching, is a wonderful moisturizing agent and
penetrant, is a natural hypo-allergic and has about the
same pH as skin and has recently proven to stimulate
the body’s immune system. [3,4] Plants containing Av
have been used as anti-inflammatory agents, for the
treatment of ulcer, hepatitis and neoplasms, and also
for wound healing. Recently Av preparations have
been applied for oral mucosal diseases like lichen
planus, geographic tongue, oral candidosis, and even
potentially malignant disorders.[1,5-9] The microculture
tetrazolium technique (MTT) is a quantitative, sensitive
test with which a linear relationship between cell
activity and absorbance, and hence, the cell growth or
cell death rate can be measured.

Address for correspondence: Dr. Devi Gopakumar, Department of Oral Medicine and Radiology, Noorul Islam Dental College,
Trivandrum, Kerala, India. E-mail: deviomr@gmail.com
Received: 12-08-2014  Accepted: 18-03-2015  Published: 22-04-2015

364 Journal of Indian Academy of Oral Medicine & Radiology | Oct-Dec 2014 | Vol 26 | Issue 4
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Gopakumar D and Nair SS: Aloe vera is non-toxic to cells
Materials and Methods
Fresh leaves of Av were collected from an agricultural
farm, in Trivandrum, Kerala, India. The collected Av
leaves were thoroughly washed with tap water. The gel
extracts of Av were pressed out of the leaves into a beaker
by manually squeezing the leaves. Freshly prepared Av
gel (70%) was used for the study. The dilution was done
with distilled water. L929 fibroblast cells purchased
from National Centre for Cell Sciences (NCCS), Pune,
India, was maintained in Dulbecco’s modified eagles
media and grown to confluency at 37°C and 5% CO2
in a humidified atmosphere in a CO2 incubator. L929
fibroblast cells are the cells commonly used in the cell
viability studies. Epithelial cell lineage could not survive
the study period. The cells were trypsinized using 500 µl
of 0.025% trypsin in phosphate buffer solution (PBS)/
ethylenediaminetetraacetic acid (EDTA) solution for
2 minutes and passaged to T flasks in complete aseptic
conditions and incubated. One microliter, 5 µl and 10 µl of
70% Av were added to 80% confluent cells and incubated
for 24 hours. The cytotoxic effects of medication was
determined by MTT cell viability assay.[10] The cell viability
was assessed at 540 nm at different sample concentrations.
Microculture tetrazolium technique colorimetric assay
Microculture tetrazolium technique colorimetric assay
measures the reduction of yellow 3-(4,5-dimethythiazol-2-yl)-
2,5-diphenyltetrazolium bromide (DDB) by mitochondrial
succinate dehydrogenase. 3-(4,5-dimethythiazol-2-yl)-2,5-
diphenyltetrazolium bromide enters the cells and is reduced
to an insoluble, colored (dark purple) formazan product in
the mitochondria. An organic solvent like isopropanol is
used to solubilize the cells and spectrophotometry is used
to measure the released, solubilized formazan reagent.
The viability of the cells can be measured by the level of
this activity, since reduction of DDB can occur only in
metabolically active cells.
The cell culture suspension was washed with 1x PBS
and 200 µl MTT solution was added to it (MTT-5 mg/
volume dissolved in PBS). Then it was incubated at
37°C for 3 hours. All the MTT was removed by washing
with 1x PBS and 300 µl dimethyl sulfoxide (DMSO) was
added to each culture. Incubation was done at room
temperature for 30 minutes until the cells get lysed and
a dark purple color was obtained. The solution was
transferred to centrifuge tubes and centrifuged at top
speed for 2 minutes to precipitate the cell debris. Optical
density (OD) was read at 540 nm using DMSO as blank.[10]
Results
Phase contrast analysis of L929 cell lines treated with
extracts was done with Olympus CKX41 (20X) which
Journal of Indian Academy of Oral Medicine & Radiology | Oct-Dec 2014 | Vol 26 | Issue 4
showed little or no significant changes in the cell
morphology with increased concentration [Figure 1].
Sample concentrations of 1 μl, 5 μl and 10 μl were
used in this study. The viability of cells was assessed
at 540 nm. The control cells used in the study showed
100% viability. The sample at 1 μl concentration showed
a percentage viability of 92.82 ± 2.18, 89.83 ± 3.12 at 5 μl
concentration, and 88.78 ± 1.96 at 10 μl. The results are
shown in Table 1 and Graph 1.
Discussion
The five species of aloe with much documented medical
uses include Aloe barbadensis miller, Aloe perryi baker,
Aloe ferox, Aloe arborescens, and Aloe saponaria. Their
products have been used for medical and preservative
purposes.[1,7,8] The ingredients of the plant leaf include
the sap, the mucilage and the parenchymatous gel. Its
average pH is 4.55. Gel form of Av is mostly preferred.
Its chemical and therapeutic properties have been
investigated by many studies [Table 2].[1-5]
The contents are lignin, saponins, anthraquinones and
derivatives (aloin, barbaloin, isobarbaloin, anthranol,
anthracene, loetic acid, emodin, aloe emodin, ester
of cinnamonic acid, etheal oil, chrysophanic acid,
resistannol), minerals (calcium, magnesium, sodium,
zinc, iron, manganese, potassium, copper and
chromium), vitamins (B1, B2, B6, B12, C, folic acid,
beta-carotene, E, choline and niacinamide), enzymes
(peroxidase, cellulase, aliiase, carboxypeptidase,
catalase, amylase, lipase and alkaline phosphatase),
sugars, sterols (cholesterol, campesterol, β-sitosterol
and lupeol). [3-5,8] Aloe vera contains anthraquinone,
polysaccharide and carbohydrate. Anthraquinone
is extracted from the plant before use. It has been
Table 1: Results of the MTT assay
Sample concentration OD (540 nm) Viability (%)
Control 0.669 100
1 μl 0.621 92.82±2.18
5 μl 0.601 89.83±3.12
10 μl 0.594 88.78±1.96
Figure 1: Phase contrast analysis of L929 cell lines treated with extracts
showing little or no significant changes in cell morphology with increased
concentration (Olympus CKX41, 20X)
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Gopakumar D and Nair SS: Aloe vera is non-toxic to cells

reported that it stimulates macrophages and has
antiviral effects.[4,7,8]
The presence of free radicals in the body induces cell
and tissue damage.[11,12] This sort of damage is known
as oxidative damage. The mechanism of protection
against oxidative stress can either be the decreased
production of free radical derivatives or due to the
antioxidant activity of phenolic component present
in the Av leaf gel or a cumulative effect of phenolic
component, tocopherol and other vitamins, along with
the components which are responsible for increasing
the bioavailability of the vitamins.[13,14] The antioxidants
work synergistically to neutralize free radicals, especially
vitamin E, carotene, vitamin C, and other bioflavonoids.
Tissues with high turnover rate require a rich and ready
supply of building nutrients to produce and maintain
healthy and efficient cell lineage.[12-14] Several studies
have described the antigenotoxic and chemopreventive
effects of Av. Also new vessel formation in cingulated
cortex and septal areas in rats after ischemia/reperfusion
injury (angiogenic effect) has been observed with Av
gel.[15] Some studies have also reported the depressive
effects on neurotransmission, blocking formation of
axonal reflex, anti-inflammatory and analgesic effects of
Av.[16-18] The study on anticancer effect of Av in sarcoma
by Joeng et al. showed inhibition of human cancer cells
significantly at high concentrations.[19] Our study shows
that Av preparation (70%) is non-toxic to the fibroblast
cell lineage.

The combined effect of nutrient supply, reduced

inflammation and infection by Av leads to promotion
of new cell growth and more rapid healing. Danhof
and McAnally’s study of application of Av in human
fibroblast cell cultures produced an eight-fold increase
in their replication, over control cultures. Fibroblasts
are most important cells involved in the healing
process. They produce the collagen fibers of scar
tissue.[7]

To study the effect of a drug/chemical on cells

Graph 1: Graphical representation of sample concentration and cell
viability. On X-axis = Volume of sample (μl); On Y-axis = % viability
propagated in vitro there are various assays which
can be used.[20] The measurement of cell viability and
growth is a valuable tool in a wide range of research
areas. These assays range from simple to complex. The
simple assays measure the cell viability after drug/

Table 2: Chemical composition and properties of Av[2-5,8]

Class Compounds
Anthraquinones/anthrones Aloe-emodin, aloetic-acid, anthranol, barbaloin,
isobarbaloin, emodin, ester of cinnamic acid
Carbohydrates Pure mannan, acetylated mannan, acetylated
glucomannan, glucogalactomannan, galactan,
galactogalacturan, arabinogalactan, galacto-
glucoarabinomannan, pectic substance, xylan, cellulose
Chromones 8-C-glusoly-(2’-O-cinnamoly)-7-O-methlyaloediol
A, 8-C-glucosyl-(S)-aloesol, 8-C-glucosyl-7-O-
methylaloediol A, 8-C-glucosyl-7-0-methylaloediol,
8-C-glucosyl-noreugenin, isoaloeresin D,
isorabaichromone, neoalosin A
Enzymes Alkaline phosphatase, amylase, bradykinase,
carboxypeptidase, catalase, cyclooxidase,
cyclooxygenase, lipase, oxidase, phosphoenolpyruvate,
carboxylase, superoxide dismutase
Inorganic compounds Calcium, chlorine, chromium, copper, iron, magnesium,
manganese, potassium, phosphorous, sodium, zinc
Proteins Lectins, lectin-like substance
Vitamins A, B12, C, E, choline and folic acid
Hormones Auxins and gibberellins
Properties
Aloin and emodin act as analgesics,
antibacterials and antivirals
Aloe single component (alprogen), a
glycoprotein has antiallergic and novel
anti-inflammatory properties
Anti-inflammatory action
Bradykinase helps to reduce excessive
inflammation when applied to the
skin topically, while others help in the
breakdown of sugars and fats
Essential for proper functioning of various
enzyme systems in different metabolic
pathways and a few are antioxidants
Lignin, enhances the penetrative effect of
the other ingredients into skin. Saponins
have cleansing and antiseptic properties
Vitamin A, C and E are antioxidants and
antioxidants neutralize free radicals
Helps in wound healing and have
anti-inflammatory action

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Gopakumar D and Nair SS: Aloe vera is non-toxic to cells
chemical exposure, an example of which is the dye
exclusion which measures the membrane integrity and
the effect of the drug on cell growth wherein the cells
are simply enumerated. Complex assays measure cell
viability by evaluating the capacity of the cell to reduce
compounds.
Some of the assays which can be employed include:
1. Trypan blue staining: It assesses the cell membrane
integrity. It is not sensitive and cannot be adapted
for high-throughput screening.
2. Uptake of radioactive substances: Usually tritium-
labeled thymidine is used. It is accurate but time-
consuming and involves handling of radioactive
substances.
3. Clonogenic assay: It is the gold standard which
measures the proliferative potential of cells. It is
more difficult and time-consuming. It measures the
effect of the compound on the proliferating fraction
of the cell population by measuring the percentage
of cells in the population capable of giving rise to
clones.[20]
In our study cell viability was determined by using
the MTT. The results were then quantified by
spectrophotometric means. For each cell type a linear
relationship between cell number and absorbance was
established, enabling accurate, quantification of changes
in cell proliferation. The applications for this method
include drug sensitivity, cytotoxicity, response to growth
factors, and cell activation.[10,21-23] The MTT assay was
utilized by Mosmann[24] to quantitate cellular growth
and cytotoxicity and subsequently was investigated by
the National Cancer Institute in 1986.[25] The MTT system
has more advantages compared to the other tests. Table 3
shows the differences between the MTT assay and trypan
blue staining.[19,26]
A study on human corneal cells showed no toxicity
of ethanol, ethyl acetate and heptane extracts of Av.
According to the study there was no reactive oxygen
species (ROS) reducing activity by the heptane
extract and trace action by the ethanol (only at a
high concentration of 125 µg/ml) extract of Av. Free
Table 3: Differences between the MTT assay and trypan blue
staining
MTT assay Trypan blue staining
Quantitative Qualitative
Can be adapted to high- Cannot be adapted for high-
throughput screening throughput screening and must be
read individually
More sensitive Less sensitive
Can also be used Can be applied only if a cell is
to study cell death alive
mediated by a
cytotoxic agent
Journal of Indian Academy of Oral Medicine & Radiology | Oct-Dec 2014 | Vol 26 | Issue 4
radical scavenging effect was seen only with the
ethyl acetate extract. Nitric oxide (NO) production
by human corneal cells decreased with plant extracts
when compared to the untreated controls. After the
addition of Av extracts to the culture media, the
cytokine (IL-1β, IL-6, TNF-α and IL-10) production
decreased.[27] A comparative study on oral submucous
fibrosis by Santosh et al. showed that the efficacy was
much better in the herbal antioxidant oxitard group
when compared to the patients who were advised to
apply Av gel locally. [28] Pubmed literature search on
studies on cytological effects of Av on human cells
revealed scarce studies. Only a few studies were
done to assess the cytotoxic effect of aloe species.
Our study shows that Av is cytofriendly, as the cells
were viable at different concentrations. Recent trends
in the use of Av in the treatment of many reactive
and potentially malignant disorders of oral cavity
should be safe, and should open a new era for the
clinical application of Av in many mucocutaneous
diseases.
Conclusion
The present study has estimated the cell viability at
different concentrations of Av concluding the non-
cytotoxic nature of the therapeutic dosage of Av on
human cells. The cytofriendly result of the commonly
used concentration (70%) of Av should prompt clinicians
to consider this eco-friendly product for treatment of
many other common oral diseases in future. The multi-
therapeutic pathways of action of Av with its non-toxic
nature should also prompt many researchers for further
molecular analysis.
Acknowledgement
We would like to acknowledge Dr. Rajesh Ramachandran,
Director, Biogenix Research Centre, Kerala, and his team for
the support in conducting this study.
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How to cite this article: Gopakumar D, Nair SS. Aloe vera is non-
toxic to cells: A microculture tetrazolium technique colorimetric assay
study. J Indian Acad Oral Med Radiol 2014;26:364-8.
Source of Support: Nil, Conflict of Interest: None declared.