Papain, a phytotherapeutic agent, has been used in the treatment of eschars and as a debriding chemical agent to remove damaged or necrotic tissue of pressure ulcers and gangrene. Its benefits in these treatments are deemed effective, since more than 5000 patients, at the public university hospital at Rio de Janeiro, Brazil, have undergone papain treatment and presented satisfactory results. Despite its extensive use, there is little information about toxic and mutagenic properties of papain.
This work evaluated the toxic and mutagenic potential of papain and its potential antioxidant activity against induced-
The belief that natural medicines are much safer than synthetic drugs has caused exceptional growth in human exposure to natural products, as plants, phytotherapeutic agents, and phytopharmaceutical products. This fact has lead to a resurgence of scientific interest in their biological effects. In most countries there is no universal regulatory system insuring the safety and activity of natural products and they had not been sufficiently investigated analytically or toxicologically [
Herbal medicines can be potentially toxic to human health. In this way, scientific research has shown that many plants used in traditional and folk medicine are potentially toxic, mutagenic, and carcinogenic [
The juice of ripe papaya displayed in vivo and in vitro activities against oxidative stress [
The green (unripe) papaya, which is rich in papain, is used for dressing of ulcers. This treatment is described as effective and it is recommended in preference to other dressings for chronic skin ulcers. It has been used in many countries such as England, Nigeria, Ghana, Gambia, India, and Jamaica [
The demonstration of the phytotherapeutic potential of a given species is a difficult task, since plant extracts consist of complex mixtures of major compounds, minor concomitant agents, and fibers, which can all be involved in the observed effects. Thus, given the difficulties in determining the contribution of a specific substance in the biological effects exerted by whole extracts, the aim of this work was the study of papain isolated from
Papain, a purified protein extracted from the latex of the unripe papaya, is widely used by Brazilian nurses in traditional medicine. It can be an alternative to green papaya and it can be used as phytotherapeutic agent in the treatment of pressure ulcers, gangrene, eschars, and as a debriding chemical agent to remove damaged or necrotic tissue [
Short-term tests have been used to check compounds for their ability to induce lesions in DNA, which may lead to genotoxicity, cytotoxicity, or mutagenicity. The experimental techniques using microbial cells such as
Hydrogen peroxide (H2O2) is a normal cell metabolite formed in several enzymatic and nonenzymatic reactions. H2O2 leads to oxidative stress, mutagenicity, loss of cell function, and ultimately apoptosis or necrosis [
It is well documented that oxidative damage has been implicated in various systemic chronic diseases such as cancer, Alzheimer's disease, rheumatoid arthritis, cardiovascular disease, cataracts, and other ageing processes. Reactive oxygen species (ROSs) are essential intermediates in oxidative metabolism. Nonetheless, when generated in excess, ROSs in various active forms can damage tissues [
In recent years, there has been a considerable interest in finding natural antioxidants from plant materials to replace synthetic molecules. Data from both scientific reports and laboratory studies show that plants contain a large variety of substances that possess antioxidant activity. Phytochemicals with antioxidant effects include some cinnamic acids, coumarins, diterpenes, flavonoids, lignans, monoterpenes, phenylpropanoids, tannins, and triterpenes. Natural antioxidants occur in all higher plants and in all parts of the plant (wood, bark, stems, pods, leaves, fruit, roots, flowers, pollen, and seeds) [
The present work was carried out to evaluate the potential cytotoxic and mutagenic effects of papain using
In order to prepare cultures to evaluate cytotoxic, antioxidant, mutagenic effects of papain and to perform reverse mutation test, the strains were grown according to previously described methods [
Samples (
Plasmid pUC 9.1 has been prepared according to a previously described alkaline method [
Bacto agar, bacto tryptone, and bacto yeast extract were purchased from Difco Laboratories, Detroit, USA. Nutrient broth was purchased from Oxoid do Brasil, Ltda (Brazil). Papain powder (from
Papain powder, protected against ambient light, was suspended in 0.9% NaCl sterile solution, vigorously shaken for 2 minutes at room temperature and immediately filtered through a sterile 0.22
For the tests, the concentration of papain was calculated based on the amount administered to the patients. The highest concentration of papain (500
The culture media, solutions, and cell growth were prepared as previously described [
Dilutions of the chemical preparations and bacterial cultures were carried out with 0.9% NaCl sterile solution, dimethyl sulfoxide (DMSO), or ultra pure Milli-Q water [
In order to evaluate the potential cytotoxic effect of papain, culture aliquots (1 mL) of
In order to evaluate the cytotoxic effect of papain associated with talc, aliquots (1 mL) of
In the growth inhibition test, 100
The assays were performed according to Blanco et al. (1988);
Agarose gel electrophoresis (0.8%) was performed in order to separate different structural conformations of pUC 9.1 plasmid DNA after papain treatment: form I supercoiled (SC) native conformation, form II open circle (OC) resulting from single strand breaks, and form III linear (L) resulting from double strand breaks. Plasmid DNA aliquots (200 ng) were treated with increasing concentrations of papain for 40 minutes at 25
The results were analyzed by ANOVA since (i) the data were normally distributed as verified by the method Kolmogorov and Smirnov and (ii) samples from populations had identical standard deviations (SDs), as verified by the Bartlett method. ANOVA was followed by the Student Newman Keuls multiple comparison test using the statistical program InStat version 3.01 (GraphPad Software, San Diego, CA, USA). These analyses compared the results obtained by the several treatments, at different papain concentrations, including the controls. A significance level of 5% was adopted to evaluate the data.
The data collected by densitometry provided us with null events percentage (no breaks =
In order to evaluate toxic effects of papain cytotoxicity assay was performed. The results indicated that papain was not cytotoxic to
Effect of different papain concentrations on the survival of
Papain concentrations ( | AB1157 (WT) | BW9091 ( | BH20 ( | PQ65 ( |
50 | 0.81 ± 0.04 | 1.00 ± 0.06 | 1.10 ± 0.05 | 0.98 ± 0.04 |
250 | 1.24 ± 0.08 | 1.40 ± 0.04 | 0.80 ± 0.02 | 1.04 ± 0.01 |
500 | 0.80 ± 0.06 | 1.10 ± 0.03 | 0.90 ± 0.04 | 0.93 ± 0.02 |
Negative control 0.9% NaCl (50 | 1.00 ± 0.02 | 1.00 ± 0.05 | 0.90 ± 0.03 | 1.10 ± 0.04 |
Positive control H2O2 (10 mM) | 0.20* ± 0.01 | 0.003* ± 0.00004 | 0.17* ± 0.007 | 0.19* ± 0.01 |
The results are not significantly different
Effect of papain associated with talc on the survival of
Agents | AB1157 (WT) | BW9091 ( |
0.9% NaCl (negative control) | 1.00 ± 0.02 | 1.00 ± 0.04 |
Papain (500 | 1.00 ± 0.03 | 0.93 ± 0.03 |
Papain associated with talc (500 | 0.80 ± 0.01 | 0.96 ± 0.03 |
Talc (500 | 0.90 ± 0.01 | 0.87 ± 0.02 |
H2O2 (10 Mm) (positive control) | 0.20* ± 0.004 | 0.003* ± 0.00004 |
The results are not significantly different
This same methodoly was used to test papain in association with talc, as shown in Table
Since some reports [
Another methodology to investigate the toxic effect of papain comprised the use of Growth inhibition test. The results concerning papain potential toxic effects obtained with
Inhibition halos (mm) of the
Growth inhibition halos (mm) of | ||||||
Agents (10 | IC203 | IC204 | IC205 | IC206 | IC207 | WP2 (WT) |
H2O2 (300 | 48.0 ± 1.4 | 30.5 ± 2.7 | 20.0 ± 2.4 | 17.0 ± 2.3 | 37.7 ± 1.0 | 16.0 ± 0.5 |
Papain (100 | 45.0 ± 0.1* | 22.0 ± 1.8* | 21.5 ± 1.8 | 16.5 ± 1.7 | 33.5 ± 1.0** | 15.6 ± 0.4 |
Papain (500 | 40.5 ± 0.9* | 22.0 ± 1.9* | 20.0 ± 2.2 | 17.5 ± 2.0 | 35.5 ± 0.5** | 15.4 ± 0.8 |
0.9% NaCl (negative control) | ND | ND | ND | ND | ND | ND |
ND: not detected.
The results obtained with WP2 Mutoxitest showed that all the tested papain concentrations did not present mutagenic activity with
Mutoxitest—Number of Trp+ revertants/plate (mean ± SD). Aliquots (100
Number of Trp+ revertants/plate (mean ± SD) | ||
Agent ( | ||
Papain 5 | 158 ± 26.0 | 15 ± 3.0 |
Papain 25 | 162 ± 16.0 | 17 ± 3.0 |
Papain 50 | 154 ± 26.0 | 12 ± 3.0 |
Papain 100 | 129 ± 24.0 | 14 ± 3.0 |
Papain 125 | 178 ± 10.0 | 16 ± 3.0 |
Papain 250 | 162 ± 25.0 | 17 ± 3.0 |
Papain 500 | 149 ± 24.0 | 18 ± 3.0 |
Positive control H2O2 (300) | 853* ± 70.2 | 15a ± 1.9 |
Negative control 0.9% NaCl (50 | 141 ± 28.0 | 16 ± 4.0 |
Conformational changes in plasmid DNA (pUC 9.1) after treatment with different concentrations of papain were also investigated, using agarose gel electrophoresis analysis. Data showed (Figure
Analysis of plasmid pUC 9.1 DNA strand breaks after treatment with papain. Aliquots of pUC 9.1 plasmid DNA (200 ng) were incubated with different concentrations of papain for 40 minutes at 25
In patients from a Brazilian university public hospital (Pedro Ernesto Hospital), papain, associated or not with talc, is used for the topical treatment of chronic skin ulcer. It is described as effective and recommended in preference to other dressings for the same purpose, as unripe papaya, in other countries [
Despite its extensive use in Brazilian patients, there is still little information about papain toxicity. Then
The phytotherapeutic agent papain was not able to induce inactivation of all the
Even
DNA repair deficient strains were also resistant to papain treatment (Table
The antioxidant activity of papain against H2O2-induced damage was also assessed using Growth inhibition test. When the cells were simultaneously treated with H2O2 (300
Mutoxitest is a potent assay to assess the ability of a series of compounds to induce reversion of the trpE65 mutation in
Circular plasmid DNA was used in vitro as target to study the induction of strand breaks in DNA by compounds such as oxidant agents and natural products [
Our study indicates that papain is not toxic and/or mutagenic in bacterial systems. Indeed, papain revealed to be an antioxidant agent against H2O2-induced damage.
Webman and coworkers (1989) and Mehdipour and coworkers (2006) demonstrated an antioxidant effect of ripe
A problematic aspect in understanding potential toxicological events relevant to the medicinal use of
Oloyede (2005) studied unripe pulp of
Our results further support the notion that papain, the compound isolated from latex of unripe
The authors gratefully acknowledge the following institutions and members of (1)