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 Table of Contents  
ORIGINAL ARTICLE
Year : 2014  |  Volume : 26  |  Issue : 4  |  Page : 364-368

Aloe vera is non-toxic to cells: A microculture tetrazolium technique colorimetric assay study


1 Department of Oral Medicine and Radiology, Noorul Islam Dental College, Trivandrum, India
2 Department of Oral and Maxillofacial Pathology, Pushpagiri College of Dental Sciences, Tiruvalla, Kerala, India

Date of Submission12-Aug-2014
Date of Acceptance18-Mar-2015
Date of Web Publication22-Apr-2015

Correspondence Address:
Devi Gopakumar
Department of Oral Medicine and Radiology, Noorul Islam Dental College, Trivandrum, Kerala
India
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Source of Support: None, Conflict of Interest: None


DOI: 10.4103/0972-1363.155628

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   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.

Keywords: Aloe vera , colorimetric assay, cytotoxicity, microculture tetrazolium technique (MTT) assay


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

How to cite this URL:
Gopakumar D, Nair SS. Aloe vera is non-toxic to cells: A microculture tetrazolium technique colorimetric assay study. J Indian Acad Oral Med Radiol [serial online] 2014 [cited 2020 Aug 5];26:364-8. Available from: http://www.jiaomr.in/text.asp?2014/26/4/364/155628


   Introduction Top


Thomas 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 been used for an array of ailments since ancient times as a medicinal plant. There are more than 360 different 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],[6],[7],[8],[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.


   Materials and Methods Top


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% CO 2 in a humidified atmosphere in a CO 2 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 Top


Phase contrast analysis of L929 cell lines treated with extracts was done with Olympus CKX41 (20X) which 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 [Additional file 1] ].{Figure 1}{Table 1}


   Discussion Top


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],[2],[3],[4],[5]{Table 2}

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],[4],[5],[8] Aloe vera contains anthraquinone, polysaccharide and carbohydrate. Anthraquinone is extracted from the plant before use. It has been 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],[13],[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],[17],[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 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/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:

Trypan blue staining: It assesses the cell membrane integrity. It is not sensitive and cannot be adapted for high-throughput screening.

Uptake of radioactive substances: Usually tritium-labeled thymidine is used. It is accurate but time-consuming and involves handling of radioactive substances.

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],[22],[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]{Table 3}

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 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 Top


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 Top


We would like to acknowledge Dr. Rajesh Ramachandran, Director, Biogenix Research Centre, Kerala, and his team for the support in conducting this study.

 
   References Top

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Friedman RN, Si K. Initial characterization of the effects of Aloe vera at a crayfish neuromuscular junction. Phytother Res 1999;13:580-3.  Back to cited text no. 16
    
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Kim HS, Kacew S, Lee BM. In vitro chemopreventive effects of plant polysaccharides (Aloe barbadensis Miller, Lentinus edodes, Ganoderma lucidum and Coriolus versicolor). Carcinogenesis 1999;20:1637-40.  Back to cited text no. 18
    
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Esua MF, Rauwald JW. Novel bioactive maloyl glucans from Aloe vera gel: Isolation, structural elucidation and in vitro bioassays. Carbohydr Res 2006;341:355-64.  Back to cited text no. 20
    
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Cory AH, Owen TC, Barltrop JA, Cory JG. Use of an aqueous tetrazolium/formazan assay for cell growth assays in culture. Cancer Commun 1991;3:207-12.  Back to cited text no. 21
    
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Slater TF, Sawyer B, Straeuli U. Studies on succinate-tetrazolium reductase systems. III. Points of coupling of four different tetrazolium salts. Biochim Biophys Acta 1963;77:383-93.  Back to cited text no. 22
    
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28.
Patil S, Halgatti V, Maheshwari S, Santosh BS. Comparative study of the efficacy of herbal antioxdants oxitard and Aloe vera in the treatment of oral submucous fibrosis. J Clin Exp Dent 2014;6:e265-70.  Back to cited text no. 28
    


    Figures

  [Figure 1]JIndianAcadOralMedRadiol_2014_26_4_364_155628_f1.jpg
 
 
    Tables

  [Table 1]JIndianAcadOralMedRadiol_2014_26_4_364_155628_t2.jpg, [Table 2]JIndianAcadOralMedRadiol_2014_26_4_364_155628_t3.jpg, [Table 3]JIndianAcadOralMedRadiol_2014_26_4_364_155628_t4.jpg



 

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