Introduction

Carica papaya is one of the many traditional plants widely used in folk medicine for treatment of various ailments. Its seed is althelminthic and used as vermifuge in India¹, Central and South America²  and even throughout the world³. The leaf extracts are also proved to be anti-malarial in animal models with more efficacy against Plasmodium berghei in mice when compared with the efficacy of the bark of Alstonia broonai⁴. Its fruit and seed extracts have pronounced antibacterial⁵, anti-amoebic⁶ activities  as do its milk latex⁷. In view of its wide application attempt has been made to ascertain whether its seed extract is effective against yet another protozoal causative agent of animal disease i.e. the trypanosomes.  In the current study, the anti-trypanosomal activity of the aqueous extracts of the seeds against the development of a protozoal parasite Trypanosoma evansi in mice was investigated.

Materials and Methods

Experimental animals

Six-week old  male ICR mice (body weight (BW) ranged 25-30mg) obtained from the Animal House, Universiti Kebangsaan Malaysia were used in this study. The mice (5 per group) were kept in the standard propylene cages and acclimatized before the start of the experiment. They were fed with the commercial mouse pellets plus drinking water ad libitum and maintained and handled according to the Universiti Kebangsaan Malaysia Animal Ethics Committee (UKMAEC) Guidelines⁸.

Inoculation of trypanosome

Mice-maintained T. evansi from Parasitology Laboratory, School of Biosciences and Biotechnology, Faculty of Science and Technology, Universiti Kebangsaan Malaysia was used. A dose of 5.0×10³ trypanosomes per mouse prepared from infected blood and suspended in 0.1ml suspension was injected intraperitoneally (i.p.) to initiate infection.

Preparation of fresh aqueous seed extract of C. papaya and its administration into experimental animals

Seeds were harvested from fresh fruits with their mucilaginous coating removed and then processed to obtain aqueous extracts according to Kermanshai et 2001⁹.  The extracts were prepared by grinding the seeds with a ground mortar and pestle using dH₂O (1g seeds in 5mL dH₂O water) at room temperature and sieved using laboratory test sieve to remove debris and stirred to homogenize before storage at 4⁰C as the original supernatant  until used.

For the treatment of the mice, three different dosages were prepared by serial dilutions with dH₂O of the original supernatant viz. 250, 500 and 750 mg per kg body weight (BW) respectively. Each dose was contained in 0.1mL suspension and injected i.p.  into each mouse.

Evaluation of anti-trypanosomal activity/parameters

Groups of 5 mice were treated with the respective dosages of the extract either 5 days pre-infection, concurrently with the infection on day 0 or 5 days post-infection and continued daily until the mice succumbed or survived. Negative control mice were infected with the parasites only whereas positive control mice were infected with the parasites plus treatment with the standard anti-trypanosomal drug Berenil at 0.35mg per 100g BW¹⁰ .

Degree of parasitaemia (rate of  infection in the mice) was determined daily using thin blood smears prepared from tail blood stained with Giemsa`s stain and observed under light microscope at x100 magnification¹¹,^12. The same smears were also used to determine the prepatent period. The mortality or survival rate of the mice was also recorded. Any bodily physical changes of the mice and changes in the BW were also noted.

Analysis of results

The results were expressed as mean +  standard deviation (SD). Data were analyzed using Minitab Pro 16.1.0.0 and Window SPSS version 16. Values of p<0.05 was taken as significant

Results

There were no obvious changes in the physical appearance of the treated mice. The overall changes in BW for all treated groups were not significantly different (p>0.05) from one another and that the positive control group mice had slight gained in BW following recovery. Mice which succumbed showed slight reduction in their BWs.

Prepatent period and survival time

Table 1 below shows the prepatent period (day) and survival time (day) of all groups. The prepatent period is defined as the period by which the parasite first appeared in the blood following the infection. In this study it was found that irrespective the dosage of the treatment received by the mice, all groups (positive control group, negative control group, pre-, concurrent- or post-infection treated groups) showed insignificant different (p>0.05) in their prepatent periods which lasted between 2 to 3 days only.

Results showed that the negative control group had the shortest survival time (i.e. 5.0 + 0.0 days) whereas the positive control group had the longest (>15.0 days) as compared to the rest of the groups (p<0.05). The pre-infection treated group receiving 750 mg per kg BW aqueous extracts survived the longest (i.e. 11.4 + 1.34 days) amongst all the treated groups.

Patent of parasitaemia

All groups showed an upward increase in parasitaemia 3 to 4 days post- infection of T. evansi  but the patterns  varied thereafter. Negative control group had a drastic increase in parasitaemia reaching its peak of   >35% in a short time before the mice succumbed in 5 days (Table 1; Figure 1). Positive control group had a short patency and by days 6 or 7, the blood was cleared of the parasites and all mice survived until the end of the experiment.

Table 1.  Means of Prepatent Period and Survival Time of All Experimental Groups Mice Following Treatments With yhe Various Dosages of the Aqueous Extracts of C.Papaya.

^*treated with Berenil; ^** Mean + s.d.

Figure 1: Patterns of parasitaemias in all the experimental groups mice following treatment with  500mg/kg BW aqueous extract of C. papaya.   Click here to View figure

Parasitaemias in all the treated groups were, to a certain degree,  suppressed and increased at a much slower rate as compared to those in the negative control mice and the mice survived longer. The patterns of suppression of the parasitaemias were however, varied between the treated groups. The obvious suppression was observed more consistent in the two pre-infection treatment groups both receiving 500 mg and 750mg per kg BW respectively. Treatment with 500 mg per kg BW of the extract caused a greater degree of suppression than those of the post-infection and concurrent-infection treatment groups (Figure 1) with the same dose. Treatment with 750mg per kg BW of the extract caused even higher degree of suppression both in the pre- and concurrent-infection treatment groups (Figure 2). The suppression in parasitaemias caused by the pre-infection treatment with a higher dosage of  750mg per kg BW of the extracts was consistent to indicate the anti-trypanosomal activity of the aqueous extracts of C. papaya against T. evansi in mice.

Figure 2: Patterns of parasitaemias in all the experimental groups mice following treatment with  750 mg/kg BW aqueous extract of C. papaya.   Click here to View figure

Discussion

Efforts to enravel  a new candidate from plant-based sources for potential drugs as alternative to the current available drugs which normally will cause resistant for the  treatment of diseases including trypanosomiasis have long been sought. Local medicinal plants were always became the choice which include C. papaya or papaya. Anthelminthic and  anti-bacterial activities of this plant were made known as described above but the information regarding its anti-protozoal activity remained to be further elucidated. In this paper, evidence in presented to indicate that its seed extracts may also proved to be anti-protozoal as well.

The appearance of a normal physical external body conditions as observed in the extract-treated groups of mice may indicate that the extracts did not cause  adverse effects/side effects on the mice. This condition on the other hand may also prove that the aqueous extract was not toxic to the mice after all.

Decreased in BW of the infected/ or treated mice or gained in BW of the recovered mice were the common phenomena resulted from parasitic infection.  Regarding the short term prepatent periods (2-3days) of all the experimental mice as shown in this study may indicate that the parasites have equal chance to develop in their respective hots at the beginning and the presence of external materials in the host`s body such as the aqueous seed extracts may alter their fate thereon. Perhaps the concentration of the extract was still low and could not exert any effects on the parasites.

Development of parasitaemia in respective groups of mice can be related to whether the mice received treatments or not. This in turn will determine their survival times. As such the negative control mice (infected but untreated group) experienced drastic increase in parasitaemia and had the shortest survival time (5.0 + 0.0 days). Short patency in the posititive group mice survived the longest amongst all the groups was due to the efficacy of the curative drug Berenil to wipe out the parasites from peripheral blood¹⁰.

The degree of the suppression of parasitaemias in all treated groups  can be related to the time when the mice first received the treatment either pre-, concurrent with- or post-infections. Irrespective of the dosage of the extract used in this study, post-infection treatments had caused the least suppressed parasitaemia as compared to the pre- and concurrent with infection treatments. Both the post- and concurrent with infection treatments appeared to cause a greater suppression and slower rate of parasitaemia. The 750mg/kg BW treatment had a greater impact on the development of the parasites. The mice had 5 days pre-infection treatments with the extracts and then continued daily for the next 11 days post-infection might have arrested the development of the parasites in the blood and caused the mice to survive much longer although this did not guarantee total protection for the mice.  The results presented here may indicate that the aqueous extracts of C. papaya contain bioactive compound(s) which have anti-protozoal activity or perhaps some anti-trypanosomal   activity against a flagellated parasite T. evansi in mice in vivo.

Arise et al.⁴ in his study implicated that the tannins, phenolics,  saponins and flavonoids compounds found in papaya extract displayed anti-malarial activity against a malaria parasite P. berghei in mice. The aqueous extracts of peel and seed of C. papaya was also found to have rich total phenolics and antioxidant activity¹³. Tasdemir et al.¹⁴ found that one of the  flavonoids, dihydroxyflavone had promising anti-trypanosomal against T. brucei rhodesiense as well as anti-leishmanial activities tested in vitro.  Kermanshai et al ¹ also proposed that papaya seed extracts of benzyl isothiocyanate is the sole bioactive principle effective against the helminth Coenorhabditis elegans.

Trypanosomes will generally double their populations through binary fission  six-hourly^.  If the increase in the population numbers left unperturbed or unchecked, major pathological outcomes will ensue and certainly breaking down the host’s immune responses which may lead to death. Which one of the bioactive compounds or phytochemical constituents that arrest or slow down the proliferation of the parasites as observed in the present study, remained unclear.

Flavonoids and related phenolic compounds are naturally found in vegetables, fruits, medicinal plants and beverages¹⁵. These compounds were found to possess diverse biological activities as antioxidants and as preventive compounds against cancer cells¹⁶.  The anti-trypanosomal activity of the aqueous extracts of the papaya seed which slow down the proliferation of the trypanosomes may operate through a cytotoxic activity. On the other hand as antioxidants, the compounds may also cause direct damage to the trypanosome cells¹⁷.

Alkaloids are common compounds generally found in papaya. They are nitrogenous in nature and widely used as cancer therapeutic agents¹⁸ and known to interfere with cell division1¹⁹ . Saponins which also found in papaya have the ability to  cleanse and  purify the blood²⁰. These compounds may have the anti-trypanosomal activity by reducing parasite numbers in the body thus lowering parasitaemia and extending the survival time of the infected mice as observed in this study.

As conclusion the results of the present study showed that the aqueous seed extracts of C. papaya have in vivo anti-trypanosomal activity against T. evans in mice. Further study must be carried out to determine the bioactive compound(s) from this plant which possess this activity and to elucidate their mechanisms of action.  Indirectly the results also highlight the potential of C. papaya as a new source for anti-trypanosomal compound(s) in the future.

Acknowlegments

The authors wish to record their appreciation for the help and assistance provided by the School of Biosciences and Biotechnology, Faculty Science and Technology, Universiti Kebangsaan Malaysia, Bangi.

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