Abstract: Background and Objective: Drug resistant Salmonella typhi is an alarming issue in the health sector which has drawn attention of researchers to search for alternative therapeutic sources. Due to increase in resistance of Salmonella typhi to antibiotics, the efficacy of methanolic extract of fruits of Physalis angulata (PFM) in treating S. typhi infection was investigated in present study. Materials and Methods: Apparently healthy Wistar rats were orogastrically challenged with ID of S. typhi multi-antibiotic resistant (MAR) isolate. At onset of signs of illness, the infected rats were divided into three groups-the first group was treated with the minimum inhibitory concentration (MIC) of the extract (400 mg kg–1 b.wt.) the second group with minimum bactericidal concentration (MBC ) of the extract (500 mg kg–1 b.wt.) while the third group was left untreated. Haematological and histopathological studies were carried out on the blood and the vital internal organs of the rats respectively at the termination of treatment. Liver function tests were also carried out on the rats. Results: Treatment of S. typhi infected rats with PFM (400, 500 mg kg–1 b.wt.) caused the recovery of the rats between 2 and 3 days and also caused the packed cell volume (PCV) of the infected rats to increase from 30.00±0.10% to 39.00±0.50% and 41.00± 0.20%, respectively. A similar trend was observed for haemoglobin, Red Blood Cells (RBCs) and neutrophil counts. On the other hand, it caused the counts of total White Blood Cells (WBC) and lymphocyte of the S. typhi infected rats which shot up as a result of the infection to reduce considerably. Conclusion: Hence, concluded that PFM exerted therapeutic, haematinic and immunomodulatory effects on the S. typhi infected rats treated with the extract.
INTRODUCTION
Salmonella typhi is a member of a large group of bacteria which are of prominent public health impact globally1. This bacterium belongs to a family called Enterobacteriaceae, whose members are usually Gram negative bacilli resident in the gastrointestinal tract. Salmonella typhi is the aetiology of typhoid fever which is normally treated with conventional antibiotics however because of the emergence of antibiotic resistant strains, treatment of this infection has become a serious task therefore the need for alternative therapy.
Resistance displayed by microorganisms to most commercially available conventional antibiotics is not a new phenomenon. The surge in antibiotic resistance however has led to the empirical search into the exploitation of plants for medicinal use2. Most of the plants being researched on have a history of being used in folklore medicine in different parts of the world.
Physalis angulata L. is an herbaceous annual plant belonging to the family Solanaceae. It is used in folklore medicine to treat many diseases in the western part of Nigeria. It is reported to have antibacterial activity against many bacterial species3,4. The in vitro research carried out by our team also reported the antibacterial activity of the methanolic extract of the fruits of Physalis angulata L. against multiple antibiotic resistant strain of S. typhi5,6. It therefore becomes worthwhile to investigate whether the extract can be exploited as an alternative to conventional antibiotic in the treatment of induced S. typhi infection in Wistar rats as part of the search for a more effective therapy. In this study therefore, assays such as histopathology, haematology and biochemistry were used in investigating the therapeutic and immunological potentialities of the methanolic extract of the fruit of P. angulata against infections caused by S. typhi in vivo.
MATERIALS AND METHODS
Study area: This research was carried out between February-April, 2017, at the Department of Microbiology, Federal University of Technology, Akure, Ondo State, Nigeria. The animal bioassay was conducted in the Animal House of the Department of Anatomy, College of Medicine, Afe-Babalola University, Ado-Ekiti, Ekiti State, Nigeria
The Physalis angulata plant used was authenticated at the University of Lagos Herbarium with the authentication number LUH: 7660. The S. typhi used in this investigation was isolated from HIV patients in a tertiary healthcare institution in Nigeria using the method of Cheesbrough7. The isolate S. typhi (MAR) was a multi-antibiotic resistant strain but which was susceptible to PFM using agar well diffusion assay carried out during preliminary studies. The MIC and MBC of the extract were determined adopting the method of EUCAST8 and Buller et al.9 respectively. The method of Adebolu et al.10 was adopted for the animal bioassay. Acclimatization was carried out for a period of 14 days. The Infectivity dose (ID) of S. typhi was determined using fifteen Wistar rats. The rats were infected orogastrically. The determined ID (1.1×106 CFU mL–1) was administered to the rats and they were observed for a period of five days for signs of infection. The Wistar rats were divided into three therapeutic groups, the 1st group was treated with the MIC (400 mg kg–1 b.wt.), the sec group was treated with the MBC of PFM (500 mg kg–1 b.wt.) while the 3rd group was not untreated. The treatment was initiated at the establishment of infection and terminated at 120 h of administration of extract. Haematological analyses were carried out to determine the total WBC, differential WBC, RBC counts, PCV and haemoglobin, using a standard reference11. Biochemical markers were analysed by adopting the methods of Shakya et al.12 with little modifications while histopathological studies were carried out following the procedures of Slaoui and Fiette13.
Statistical analysis: Data generated were subjected to ANOVA using XL-Sat.2016 version.
RESULTS
Effect of PFM on recovery duration: The S. typhi infected rats treated with PFM (500 mg kg–1 b.wt.) recovered within two days of the commencement of treatment, while the group treated with PFM (400 mg kg–1 b.wt.) recovered within 3 day of commencement of treatment (Table 1).
Effect of PFM on PCV and other blood parameters: Treatment of S. typhi infected rats with PFM caused the PCV which reduced from 50.00±0.33% in uninfected rats to 30.00±0.10% as a result of the infection to increase to 41.00±0.20% for the rats administered the MBC of PFM (500 mg kg–1 b.wt.) and 39.00±0.50% for the rats treated with the MIC (400 mg kg–1 b.wt.) of PFM. Similar trends were observed in the haemoglobin and RBC counts (Fig. 1).
Effect of PFM on total and differential WBC counts: Treatment of S. typhi infected rats with PFM caused the WBC and lymphocyte counts which increased as a result of the infection (7.20±0.12×103 mm–3 and 60.00±0.38%, respectively) from 5.80±0.11×103 mm–3 and 40.00±0.46%, respectively on the other hand reduce to 6.40±0.35×103 mm–3 and 47.00±0.45%, respectively for those administered with PFM at 500 mg kg–1 b.wt.
| Fig. 1: | Effects of administration of PFM on the haematological parameters and weights of rats infected with S. typhi, 418A*: Subgroup infected with S. typhi but not treated (Negative control), 418B: Subgroup infected with S. typhi and treated with 500 mg kg–1 of PFM extract, 418C: Subgroup infected with S. typhi and treated with 400 mg kg–1 of PFM extract, CTh: Therapeutic control |
| Table 1: | Effect of administration of different doses of PFM on the recovery of rats infected with multi-antibiotic resistant S. typhi |
418B: Group infected with S. typhi and treated with 500 mg kg–1 of PFM extract, 418C: Group infected with S. typhi and treated with 400 mg kg–1 of PFM extract | |
These values were a little lower than the values obtained from the groups treated with 400 mg kg–1 of PFM. On the other hand, treatment with PFM caused the neutrophil counts which decreased from 50.00±0.41% to 30±0.21% as a result of the infection to increase to 43.00±0.11% for the group treated with 500 mg kg–1 of PFM and 35.00±0.20% for those treated with 400 mg kg–1 b.wt. (Fig. 1).
Effect of treatment of S. typhi infected rats with PFM on weight: Treatment with PFM also caused weight gained in the S. typhi infected rats treated with PFM (500 mg kg–1±2%, 400 mg kg–1±4%) as against the infected rats that were not administered the PFM (Fig. 1).
Effect of treatment of S. typhi infected rats with PFM on liver enzymes: Infection of rats with S. typhi caused an increase in alkaline phosphatase (ALP) enzyme levels which further increased with treatment with PFM (Fig. 2). On the other hand, treatment of S. typhi infected rats with PFM caused a decrease in aspartate transaminase (AST) levels which shot up as a result of the infection in the rats (Fig. 3).
| Fig. 2: | Effects of administration of methanolic extract of fruits of Physalis angulata (PFM) on alkaline phosphatase levels of rats infected with S. typhi |
| 1: Infected with S. typhi and treated with 400 mg kg–1 b.wt., of PFM, 2: Infected with S. typhi and treated with 500 mg kg–1 b.wt., of PFM, 3: Rat group not infected and not treated with PFM, 4: Infected with S. typhi and treated with PFM | |
| Fig. 3: | Effects of administration of methanolic extract of fruits of Physalis angulata(PFM) on Aspartate transaminase levels of rats infected with S. typhi |
1: Infected with S. typhi and treated with 400 mg kg–1 b.wt., of PFM, 2: Infected with S. typhi and treated with 500 mg kg–1 b.wt., of PFM, 3: Rat group not infected and not treated with PFM (Control group 1), 4: Infected with S. typhi and not treated with PFM (Control group 2) |
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| Fig. 4: | Effects of administration of methanolic extract of fruits of Physalis angulata (PFM) on Alanine Transaminase levels of rats infected with S. typhi |
1: Infected with S. typhi and treated with 400 mg g–1 b.w.t., of PFM, 2: Infected with S. typhi and treated with 500 mg kg–1 b.w.t., of PFM, 3: Rat group not infected and not treated with PFM (Control group 1), 4: Infected with S. typhi and not treated with PFM (Control group 2) |
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| Fig. 5(a-d): | Photomicrograph of the (a) Liver of a rat infected with S. typhi but not treated with PFM. Haemorrhage occurs at the central vein (CV), (b) Liver tissue of control rat (Not infected and not treated), (c) Section of a liver from a rat infected with S. typhi that is treated with 400 mg kg–1 of PFM. Congested central vein (CCV) and lymphocytosis (arrow) were observed. The hepatocytes (H) appear normal and (d) Liver sample from a rat infected with S. typhi and treated with 500 mg kg–1 of PFM. Hepatocellular drainage is notable revealing recovery (arrow), though with a little congested blood vessel (CBV), dilated sinusoids (DS) and hepatocytes appear normal |
| (X400 Haematoxylin and Eosin staining technique) | |
Treatment of the infection with PFM however also caused an increase in the alanine transaminase (ALT) concentration levels which was way higher than that of the healthy control rats (75.8 U L–1±49.2) (Fig. 4).
Effect of treatment of S. typhi infected rats with PFM on internal organs: Histopathological studies of the liver of one of the S. typhi infected rats not treated with PFM revealed haemorrhage at the central vein (Fig. 5a). However, with treatment of the infected rats with PFM, the liver cells appeared normal though there was congestion in the central vein and lymphocytosis suggesting the stimulation of lymphocytes at the site of infection for administered PFM dose of 400 mg kg–1 of PFM but the liver of the group treated with 500 mg kg–1 of PFM showed a better recovery because there was notable hepatocellular drainage and no lymphocytosis (Fig. 5b-d). Acute tubular necrosis was observed in the glomerulus of the kidney of the S. typhi infected rats not administered PFM (Fig. 6a). Treatment with PFM (400 mg kg–1 b.wt.) resulted in mild interstitial inflammatory cells infiltration in the kidney while treatment with 500 mg kg–1 of PFM revealed the appearance of new cells and clearing off of dot necrosis (Fig. 6b-d).
DISCUSSION
The S. typhi infected rats treated with the higher concentration of PFM (500 mg kg–1 b.wt.) recovered faster (2 days) than their counterparts that were treated with 400 mg kg–1 b.wt. (3 days). This points to the fact that the MBC of PFM achieved the elimination of the infection faster than the MIC of the extract. The haematological parameters also attest to the dose dependent recovery pattern recorded in this study. This is in agreement to the findings of Anisuzzman et al.14.
Lymphocytosis and neutropenia that were observed in the S. typhi infected rats showed that the rats actually became sick however the administration of PFM to the infected rats caused a reversal of these effects showing that PFM has an immunomodulatory effect with a superior effect with the higher dose of PFM at 500 mg kg–1 b.wt.
| Fig. 6(a-d): | Photomicrograph of the (a) Kidney of rat infected with S. typhi without treatment with PFM. Renal tissue composed of the renal corpuscle reveals nephrotoxic acute tubular necrosis in the glomerulus, (b) Kidney tissue of control rat (Not infected and not treated with PFM), (c) Kidney of rat infected with S. typhi and treated with 400 mg kg–1 PFM. Symmetric medial hypertrophy (arrow), mild interstitial inflammatory cells infiltration (circle) and (d) Kidney of a rat infected with S. typhi and treated with 500 mg kg–1 PFM. New cells appear and there is reduction in the appearance of dot necrosis |
| a, c and d: X400 Haematoxylin and Eosin staining technique, b: X100 Haematoxylin and Eosin staining technique | |
The increase in PCV observed in the blood of S. typhi infected rats that were administered PFM shows that PFM also possess haematopoietic properties which hastened the production of RBCs which took care of the reduction which occurred during active infection of rats with S. typhi. This is in line with a report from Munshi and Montgomerry15. The weight gain at the end of the study signifies recovery in the rats administered PFMs. This is in line with findings from Kumar and Samaras16.
The observation that the administration of PFM caused an increase in the level of the enzyme ALP in the serum of S. typhi infected rats treated with the extract is indicative of the fact that PFM exerts cytotoxic effect as with the infection with S. typhi on the liver of the rats leading to high production of this enzyme. High levels of ALP are indicative of liver disease17. On the other hand, the AST values of rats infected with S. typhi and treated with PFM were observed to be almost the same as for the apparently healthy control rats which shows that PFM had little or no negative effects on the elevated AST enzyme in the serum of the rats. AST is reputed to rise upon damage to liver tissues, whereas it reverts quickly after the damage is being corrected. This corroborates the report of Shakya et al.12.
Haemorrhage occurred at the central vein in the examined section of the liver sample from one of the rats infected with S. typhi but not treated with PFM. Lesions in the liver are peculiar to the advanced state of infection with S. typhi. This was however reversed after treatment with PFM in this study. The rat group treated with 500 mg kg–1 b. wt., of PFM after infection with S. typhi experienced a significant improvement with regards to the hepatocellular drainage of the venous blood to return into circulation compared to the representative from the group treated with 400 mg kg–1 b.wt., of PFM. This also points to the dose-dependent hepatoprotective and therapeutic efficacy of the PFM against S. typhi in the liver of the rats and indicative of recovery. This tallies with the finding of Germano et al.18. For rats treated with 500 mg kg–1 b.wt. of PFM extract, new cells were developing with marked disappearance of necrosis. This shows that PFM has a stimulatory effect leading to the activation of M2 macrophages towards the recovery and stability of the renal tissues19.
CONCLUSION
This research has logically investigated the efficacy of Physalis angulata L. as a medicinal plant against S. typhi infection in vivo using the animal model. The study revealed that PFM at the dose of 400-500 mg kg–1 b.wt., exerted therapeutic activity in rats infected with S. typhi reducing the duration of infection to between 2 and 3 days with the higher dose exerting a greater effect. PFM also has haematopoietic potentials which can be exploited in treating anaemia.
SIGNIFICANCE STATEMENT
This study reports the therapeutic activity of methanolic extract of fruits of Physalis angulata based on the MIC and MBC of the extract in S. typhi infected Wistar albino rats. This study has also evaluated an aspect of the biosafety of the extract which is dose-dependent. More research works however are required before the extract could be adopted for human use. Especially in terms of dosaging, drug kinetics and biosafety.