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1From the Facultad de Medicina, the 2Departamento de Farmacia, and the 3Posgrado Interfacultades de Biotecnologia, Universidad Nacional de Colombia, Ciudad Universitaria, Bogotá, Colombia.
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Abstract |
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METHODS. A novel rabbit eye inflammation model was developed for studying the juice’s anti-inflammatory activity (based on an adaptation of the Draize test). Cytostatic activity was evaluated by measuring and comparing growth rates of cultured fibroblasts exposed and not exposed to various fruit juice concentrations.
RESULTS. P. peruviana fruit juice exhibited a mild anti-inflammatory activity compared with methylprednisolone, a known anti-inflammatory drug. An interesting dose-dependent cytostatic effect on cultured fibroblasts was also established.
CONCLUSIONS. The data found suggest that the P. peruviana fruit juice anti-pterygium effect described in traditional medicine may be related to its inhibiting fibroblast growth. The present study contributes to the pharmacologic knowledge regarding a remedy commonly used in Colombian traditional medicine.
Physalis peruviana (cape gooseberry or uchuva, as it is locally known) is a plant with multiple traditional medicinal uses. This fruit juice is used in Colombia for treating pterygium by applying it directly to the eye.8 Different active agents have been extracted from the plant, with a variety of pharmacologic activities, anti-inflammatory and cytostatic activity being the most important.9 10 11 12 Its possible beneficial effect in treating pterygium could be supported by these two activities, since inflammation and proliferation are implicated in disease progression.3 In this work, P. peruviana fruit juice’s anti-inflammatory and cytostatic effects were studied in a novel rabbit eye inflammation model. The inflammation component was evaluated by applying a modification of the Draize test. To assess cytostatic activity, we developed growth curves of primary fibroblast cultures, supplemented with or without various fruit juice concentrations and performed histologic analyses of the eyes.
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Methods |
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Vegetal Material
Fresh P. peruviana fruit, supplied by Frutifresca Ltda. (Bogota, Colombia) were macerated in a porcelain mortar until becoming pulp. An orangish, very dense and sticky liquid (pH 3), with high solid particle and sugar content was obtained from the macerated material. It was used in the animal experiments without any other manipulation, in that it had been recently extracted. For the experiments performed with primary fibroblast cultures isolated from rabbit oral mucosa, fresh juice from 500 g macerated fruit was centrifuged at 5000g for 10 minutes to eliminate most solids, filtered, and then subjected to filtration in sterile conditions in a laminar flow chamber. The sterile juice was put into amber flasks and stored at 4°C until needed for the cultures.
Draize Test Modification
Male New Zealand albino rabbits supplied by Cunicultura Bachué (Madrid, Cundinamarca, Colombia) weighing 1500 to 2000 g were used in these experiments. The animals underwent a period of adjustment for 4 days on arrival and were individually housed in stainless steel cages (40 x 40 x 50 cm), with freely available water and food concentrate (Conejina; Purina, Bogota, Colombia). The animal experiments were performed according to the ARVO Statement for the Use of Animals in Ophthalmic and Vision Research and the Colombian Ministry of Health Resolution 8084/1993. Protocols involved bilateral ocular procedures to reduce the number of animals included in the study and to diminish interanimal variability. The study was approved by the Ethics Committee of the National University of Colombia. Euthanatization was performed according to the protocols laid down in the "Report of the AVMA Panel on Euthanasia."
We initially decided to determine whether the juice of the P. peruviana fruit exhibits irritant activity by applying the Draize test.13 In this test, 0.1 mL of the substances tested were applied to the lower conjunctival sac of a rabbit’s eye, leaving the other eye as control. After 1, 24, 48, and 72 hours, alterations produced in the cornea, iris, and conjunctiva are evaluated. These alterations are graduated according to a previously established scale,13 and the obtained values were added. The sum of the two scores corresponding to the cornea (opacity and area) and one to the iris (hyperemia) were multiplied by 5, and the sum of the three conjunctiva scores (edema, hyperemia and discharge) was multiplied by 2; all these values are then added together. A category (mild, moderate, or severe irritant) was assigned to the maximum value obtained during the observations made at the established times. In the present work, once it had been proved that the juice was not an irritant, this test was adapted for evaluating the fruit juice’s anti-inflammatory activity. Such adaptation implied choosing the anti-inflammatory agent and the inflammation-inducing substance; vehicle was used as control in all cases (normal saline solution). The anti-inflammatory agents evaluated were 1% ophthalmic prednisolone and 6% methylprednisolone in parenteral presentation. As inflammation-inducing substances N,N-dimethylformamide and acetic acid were used. Observations were performed 1, 24, 48, and 72 hours and 1 and 2 weeks after applying the inflammation inducers. The alterations produced in the cornea, iris, and conjunctiva were evaluated, without washing the substance off. For the Draize test modification, three rabbits were used in each experiment. Contrary to the method used in the Draize test, we decided to sum all the scores obtained for each parameter (cornea, iris, and conjunctiva) to calculate the inflammation scores, without performing differential multiplication operations between the cornea, iris, and conjunctiva and without averaging the results obtained for the three rabbits.
Once the model had been adapted, the juice’s anti-inflammatory activity was evaluated in two groups, each of seven animals. The anti-inflammatory activity of the fruit juice was compared to that of the vehicle in one of the groups. In the other group, the fruit juice’s anti-inflammatory activity was compared to that of the chosen anti-inflammatory agent. In the first group, 0.1 mL of vehicle was placed in the right eye and 0.1 mL juice in the left eye. Twenty-four hours after application, 0.1 mL 5% acetic acid was instilled into each eye to induce injury. The same amount of vehicle or juice was added 3, 24, 48, and 72 hours after injury. Clinical evaluation of ocular inflammation was performed at 1, 24, 48, and 72 hours and 1 and 2 weeks. The same experiment was performed on the remaining group of rabbits, but substituting vehicle for 6% methylprednisolone. Induction of injury, application of the treatments, and the evaluations were performed by the same person in all cases.
Developing the Pterygium Model in Rabbits
Two different methodologies were used for inducing pterygium formation in the rabbits’ eyes. The first was based on transplanting an auricular cartilage fragment (4 x 2 mm) obtained from the same rabbit’s ear. The fragment was grafted onto the upper limbal area of the rabbit’s eye, positioning it between the conjunctiva and the sclera, by a pocket made in this space from an incision performed in the conjunctiva lower down than the site where the transplanted cartilage was.
The second methodology involved inducing a thermal injury with a soldering iron, in the limbus and an area adjacent to it. These lesions exhibited a rhomboid shape, involving an area of the cornea and an area of the conjunctiva equal to both sides of the limbus (Fig. 1) . The latter method allowed pseudopterygia to develop and was thus used in evaluating the effect of the plant’s juice in the study. Equal areas of thermal injury were created in both eyes. P. peruviana fruit juice (0.1 mL) and vehicle (0.1 mL) were applied in the left and the right eyes, respectively. An equal dose was applied once a day for a month. In the other animal group, 0.1 mL 6% methylprednisolone was applied in the right eye and 0.1 mL juice in the left one.
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Evaluating the Cytostatic Activity of the P. peruviana Fruit Juice
Primary cultures of fibroblasts isolated from rabbits’ oral mucosa according to the method described by Pérez et al.14 were used for evaluating the juice’s cytostatic effect. Cultures were incubated (5% CO2 at 37°C) with Dulbecco’s modified Eagle’s medium (DMEM; Invitrogen, Grand Island, NY), 10% fetal bovine serum (FBS; Sigma-Aldrich, St. Louis, MO), 1% nonessential amino acids (Invitrogen), 1% sodium pyruvate (Invitrogen), 1% vitamins (Sigma-Aldrich), 100 U/mL penicillin-100 µg/mL streptomycin, and amphotericin B (0.25 µg/mL; Invitrogen). Until the cells reached confluence, the medium was changed twice per week. Growth curves of third-passage fibroblasts were then drawn up. To produce the curves, the cultures were covered with 2 mL medium supplemented with sterile P. peruviana fruit extract in suitable quantities for obtaining a 10%, 5%, 2.5%, and 1% final extract concentration. In all the solutions made, the medium’s pH was 7.2. The cultures were then incubated for 24 hours at 37°C and 5% CO2. The effect of each of these concentrations was evaluated in independent dishes; a dish was thus seeded for each concentration with an additional dish to which no extract was added, serving as the control. Once incubation time had elapsed, the cells were separated from the surface of the plate with 0.25% trypsin/0.02% EDTA, collected in 15-mL Falcon tubes, and treated with medium supplemented with 10% BFS to inactivate the enzyme. Centrifuging was performed at 1800g, and the cell pellet obtained was suspended in fresh medium. The cellular suspension was treated with erythrosine B vital dye (ICN Biomedicals Inc., Costa Mesa, CA), and the cells were counted, in duplicate, in a Neubauer chamber. The foregoing procedure was applied to cultures at 0, 2, 4, 7, 9, 11, 14, 16, 18, 20, 22, 24, and 26 days. The logarithm for average cells counted in each of the wells was plotted according to elapsed culture time. The number of generations produced in relation to the initial number of cells was calculated from the results obtained, as was the time for the cell population to double.
Statistical Analysis
A Friedman nonparametric test was used to establish the significance of differences between the two groups of rabbits used to evaluate the juice’s anti-inflammatory activity; P < 0.05 was considered to be statistically significant. In all cases comparisons between treatments were blocked by tissue.
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Evaluating the Anti-inflammatory Activity of P. peruviana Fruit Juice
The irritation produced by the fruit juice on a rabbit’s eye was initially evaluated by using a Draize test.13 The average maximum score obtained was 0.5. The juice was thus classified as being nonirritant and its anti-inflammatory activity was then evaluated by modified Draize test. It was found that inflammation was less in the eyes to which the juice had been applied than that observed in the eyes treated with saline solution. Total inflammation scores were 281 and 254, respectively (Fig. 3A ; statistically significant at P = 0.014). The data from juice and methylprednisolone (6%) anti-inflammatory activity comparison is presented in Figure 3B . It also showed significant differences between the two treatments (P = 0.014); the total inflammation scores were 320 and 337, respectively.
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Discussion |
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The modification made in the present work consisted of both eyes being exposed to an irritant substance and the vehicle being added to the eye used as control and an anti-inflammatory substance to the other. This treatment was applied, because having both eyes inflamed meant that it could be established whether there were differences between the inflammation scores obtained on applying the vehicle or the anti-inflammatory substance. Inflammation was the parameter analyzed because it and fibroblast proliferation are major components of the pterygium. Acetic acid was used as an irritant substance because, after testing many substances (data not shown), we found that it could induce a moderate inflammation that resembles pterygium inflammation.
The first thing we did in adapting the test was to determine the irritant substances, the vehicle, and the commercial anti-inflammatory agents to be used. In the original Draize test, the sum of the scores corresponding to the cornea and iris is multiplied by 5, and the sum of the conjunctiva scores is multiplied by 2. All these values are then added together. A category is assigned to the maximum value obtained during the observations made at the established times. Such manipulation of scores gives a high weighted value to the scores obtained with cornea and iris, which could mean achieving 90 of the 110 points possible, meaning that the importance of the conjunctiva processes would be seen to be much reduced.16
The behavior of the inflammation in the conjunctiva should be emphasized when evaluating the anti-inflammatory activity of the juice, more than what occurred in the cornea and iris for two reasons: (1) scores corresponding to the cornea and iris evaluated inflammatory change in deeper ocular tissues, and (2) the effect of the juice was superficial, meaning that emphasis should be placed on external or superficial inflammation of the eye. We thus decided to sum all the scores obtained without giving any additional weight to any of the tissues involved, to give greater weight to the conjunctiva. Greater inflammatory reactions were presented when the inflammation-inducing substance (N,N-dimethylformamide) was added in the eyes exposed to the anti-inflammatory agent than that observed in the eye exposed to the vehicle. This result was paradoxical, as it was expected that there would have been no anti-inflammatory activity in the worst of cases, but not that there would have been greater inflammation in the eye treated with the corticoid. This result was initially attributed to the presence of benzalkonium chloride, added as preserving agent, in the ophthalmic corticoid preparation that was used. It is known that this substance can cause damage to epithelial cell membranes,17 thereby increasing inflammation already caused by N,N-dimethylformamide.
Methylprednisolone was thus evaluated in parenteral presentation according to the presumption that the ophthalmic solution’s preservative interaction with the inflammation-inducing agent was responsible for the observed inflammatory response. A higher inflammation score was also observed in this experiment in the eye in which the anti-inflammatory agent was instilled.
At this point it became evident that these results were probably not related to the substance that was being used as an anti-inflammatory agent, but rather to the substance that was being used to induce inflammation. This substance, N,N-dimethylformamide, was chosen to induce inflammation, because it is classified as a moderate inducer of irritation in the Draize test.17 It is possible that this compound affected cell membranes when it was applied in concentrated form to the mucosa, due to its great miscibility with water and organic solvents.17 Moreover, its strong solvent action could have prolonged its permanence in tissues and made its penetration much deeper. In addition, the weak penetration into the eye of topically applied anti-inflammatory compounds could have been responsible for the lack of anti-inflammatory activity observed. On the other hand, it could be attributed to the action of washing with the vehicle (normal saline solution), the least inflammation was observed in the eyes that were treated with the vehicle.
The inflammation-inducing substance was thus changed to acetic acid. This substance was chosen because it is easily obtained, and it is known that organic acids produce a concentration-dependent severity of irritation of the mucosa.17 Furthermore, low-concentration organic acids cannot penetrate tissues deeply.17 The acetic acid concentration chosen to cause superficial irritation was 5%. The Draize test continued to be adapted using methylprednisolone as the anti-inflammatory agent and, in this case, results were satisfactory (i.e., less inflammation developed in the eye treated with the anti-inflammatory).
Once the modifications had been made to the test, the inflammatory activity of P. peruviana fruit juice was then studied. A classic Draize test was first applied to the fruit juice to discount its irritant action. Once it had been determined that it did not induce irritation, a modified Draize test was used to prove whether the juice showed greater anti-inflammatory activity than that of the vehicle. Overall, our results indicate that the juice has greater anti-inflammatory activity, indeed. It should be mentioned that the anti-inflammatory activity was less than that exhibited by methylprednisolone.
P. peruviana fruit juice is used in traditional Colombian medicine in treating pterygia, therefore we tried to develop an experimental pterygium model to test it. To reach this goal, cartilage was initially implanted close to the limbus for two reasons: inducing a chronic inflammatory reaction and creating a site where there would be irregular distribution of tears over the cornea. The implantation was intended to promote fibrosis in the scleral–corneal limbus and the adjacent cornea, these being lesions characteristic of human pterygium. The results showed that the procedure did not induce development of pterygium, meaning that the hypothesis concerning irregular distribution of the lachrymal film being the origin or adjuvant in forming this type of lesions had no experimental basis.
The approach was to injure the limbus area thermally, to destroy stem cells, and to produce an advance of the conjunctiva over the cornea. This method was used to evaluate the hypothesis explaining the pathogeny of the pterygium as being secondary to destruction of scleral–corneal limbus stem cells, caused by the sun’s and wind’s prolonged action on the exposed limbus. The results obtained indicated the presence of lesions with a macroscopic appearance very similar to that of pterygium. The histologic analysis of these lesions showed a chronic inflammatory reaction and an increased number of fibroblasts and polymorphonuclear cells in the injured area. However, actinic elastosis and hyalinization of the connective tissue, the characteristic features of pterygium,3 were not seen.
When the anti-inflammatory P. peruviana fruit juice activity was evaluated in the thermally injured area, a higher degree of chronic inflammation, granulation tissue formation, vascular congestion, and fibrosis was observed in tissue treated with vehicle when compared with that treated with P. peruviana. However, comparison of the eye treated with methylprednisolone to that treated with P. peruviana showed a lesser degree of chronic inflammation, granulation tissue, slight congestion, and fibrosis. This result could be attributable to the fact that methylprednisolone is currently the most potent medicinal corticoid. Another point worth considering is that the lesions inflicted in this experiment resulted from a single acute inflammatory stimulus and that this is a very different situation to that presented during the development of pterygium, which could take several years to form. The beneficial effect of inhibiting cellular proliferation produced by the juice from P. peruviana fruit would probably be more evident in a chronic situation such as that which occurs in pterygium.
Regarding developing the pterygium model in animals, this first advance clearly showed that the induced irritation in the scleral–corneal limbus area must be chronic or at least repetitive to achieve an injury similar to human pterygium. Because of histologic findings in human pterygium showing that elastosis is related to actinic damage, an injury mechanism including chronic actinic tissue damage must also be used in inducing pterygia. A possible method would be several sessions of UV radiation applied to a particular area of the limbus, over a more prolonged period of a rabbit’s life.
The data obtained in this work show the P. peruviana extract’s cytostatic action. For example, when a 10% extract solution was used, fibroblast population doubling time was four times greater (18.9 days) than that exhibited by cells that were not exposed to it (4.7 days). Cytostatic activity was dose dependent in the range of fruit extract concentrations assayed. Although the experiments performed in this work do not allow us to state whether the cytostatic effect observed in primary cultures of fibroblasts is equivalent to in vivo P. peruviana cytostatic activity, these in vitro data suggest that its cytostatic action could be one of the reasons that it is beneficial in traditional medicine for treating pterygia. New experiments are necessary to establish this fruit juice’s cytostatic effect in vivo. Even though the animal model used in this study had its own limitations in using a model based on acute lesions for studying chronic processes, it provided the basis for developing a pterygium model and highlighting P. peruviana fruit juice’s beneficial pharmacologic activities.
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Footnotes |
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Submitted for publication May 29, 2007; revised January 2 and March 8, 2008; accepted May 19, 2008.
Disclosure: J.M. Pardo, None; M.R. Fontanilla, None; L.F. Ospina, None; L. Espinosa, None
The publication costs of this article were defrayed in part by page charge payment. This article must therefore be marked "advertisement" in accordance with 18 U.S.C. 
1734 solely to indicate this fact.
Corresponding author: Juan Manuel Pardo, Department of Surgery, School of Medicine, National University of Colombia, Ciudad Universitaria, Bogotá, DC, Colombia; jmpardom{at}unal.edu.co.
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