Evaluation of Anti-Toxoplasma activity of Basil (Ocimum basilicum) in Experimentally Infected Diabetic or Hypertensive Mice

doi:10.21203/rs.3.rs-4725679/v1

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Evaluation of Anti-Toxoplasma activity of Basil (Ocimum basilicum) in Experimentally Infected Diabetic or Hypertensive Mice

https://doi.org/10.21203/rs.3.rs-4725679/v1

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Toxoplasmosis is a cosmopolitan disease caused by an obligate intracellular protozoan parasite, Toxoplasma gondii (T. gondii). The probable correlation between toxoplasmosis and chronic disorders such as diabetes mellitus and hypertension is up challenging and still understood. Several treatments for toxoplasmosis are used but it is poorly tolerated. Herbal plant extracts are widely introduced as a possible source of treatment for several diseases. The present study aimed to assess the prophylactic and therapeutic effect of basil on toxoplasmosis in diabetic and hypertensive mice compared to spiramycin. A total number of 77 mice were divided into control, and basil treated group and the effectiveness of the drugs was measured in both hypertensive and diabetic mice. Parasitological assessment showed that all treated groups showed a statistically significant reduction in the median tissue cysts count compared to infected control groups. Combined (basil and spiramycin) showed the highest reduction rates of T. gondii cysts count in both hypertensive and diabetic groups. Histopathological assessment demonstrated moderate to marked improvement of brain, spleen and kidney inflammation in all treated groups especially the combined treated groups. In conclusion, basil showed promising an antiparasitic effect on toxoplasmosis in hypertensive and diabetic mice. More research are needed, however, to precisely investigate basil's prophylactic and therapeutic effects on chronic toxoplasmosis in both in vitro and in vivo models.

Toxoplasmosis

Basil

Herbal treatment

Diabetes

Hypertension

Toxoplasmosis is a cosmopolitan disease caused by an obligate intracellular protozoan parasite called Toxoplasma gondii (T. gondii) (Liu et al. 2015). T. gondii parasitizes many warm-blooded animals, including humans. Globally, nearly one-third of the populations are exposed to this parasite with varying degrees of severity depending on immunity (Flegr et al. 2014).

Most infections with T. gondii in healthy immunocompetent individuals are asymptomatic and self-limited (Pereira et al. 2010). Meanwhile, toxoplasmosis has been manifested as an "opportunistic disease" in immunosuppressed patients and chronic diseases causing reactivation of latent tissue cysts (Hemida et al. 2017).

Diabetes mellitus (DM) is a metabolic disease that occurs when the beta cells of the pancreas cannot produce insulin or when the body cannot effectively use the insulin that it produces. Recently, some reports have spotlighted the probable relevance of DM and infectious agents due to decreased host's immune system. A strong association has been found between toxoplasmosis and diabetes. T. gondii, has been proposed as a likely cause of DM (Majidiani et al. 2016; Abdul Lateef and Massar 2017).

Hypertension (HTN) is a risk factor for cardiovascular, and cerebrovascular diseases (Kjeldsen 2018), and it is highly prevalent across the world (Kearney et al. 2004) making HTN an important public health problem across much of the world. Numerous factors are associated with blood pressure. Research has investigated associations between the T. gondii infection and blood pressure (Erickson et al. 2022; Babekir et al. 2023). Toxoplasmosis was also reported as risk factor for HTN in diabetic patients (Han et al. 2018; Mohamed et al. 2020).

Several medical drugs have been routinely utilized in the treatment of T. gondii infection (Wei et al. 2015) such as pyrimethamine, sulfadiazine, spiramycin, or combination of drugs (Montazeri et al. 2017; Montazeri et al. 2018). These drugs mainly target the replicating tachyzoite stage (Choi and Lee 2018). Unfortunately, encysted bradyzoites are unaffected by most medications used to treat T. gondii (Barrett et al. 2019). Also, these medications may cause serious undesirable effects (Değerli et al. 2003).

For centuries, medicinal plant extracts have been utilized in treating several diseases. These herbal extracts possess a wide variety of bioactive compounds which may serve as potential alternatives to many synthetic drugs (Djeussi et al. 2013; Elazab et al. 2021).

Basil (Ocimum basilicum) (O. basilicum) is a culinary herb of the family Lamiaceae (mints) (Mostafavi et al. 2019) with variable pharmacological activities, such as anti-inflammatory, antimicrobial, antioxidant, anti-hypertensive, hypoglycemic and hypolipidemic (Rodrigues et al. 2016). Basil also showed efficient antiparasitic action against helminths (Sea et al. 2019; Osei Akoto et al. 2020), protozoa (Sanchez-Suarez et al. 2013). Recent work of its consumption has shown that basil had no genotoxic or organ toxic effects (Chandrasekaran et al. 2013).

This study aimed to evaluate the effect of basil as preventative and therapeutic agent for the treatment of toxoplasmosis among patients with hypertension and diabetes compared to standard treatment spiramycin for future development of an efficient and safe, compound with less toxicity and short medicating courses used for treating the disease.

1. Experimental animals

The present study was carried out on seventy-seven laboratory bred male Swiss albino mice 6–8 weeks old, with average weight of 20–25 gram. The animals were housed in Theodor Bilharz Research Institute (TBRI), Egypt during the period from January to April 2022. Throughout the study, the animals were kept on a standard diet containing 24% protein, 4% fat, 4–5% fiber and water ad-libitumin in TBRI. Animals were kept in suitable plastic cages at room temperature of 21 ^oC and 60% humidity.

2. Ethical considerations

The protocol of the present study was approved by Institutional Animal Care and Use Committee Beni-Suef University (BSU- IACUC) with approval number (021–140). All animals were fed, housed, and handled according to recommendations of "Guide for the care and use of laboratory animals" and were approved by a committee responsible for animal ethics concerning care for animals and safe disposal of their wastes at TBRI (National Research Council, 2010).

Mice were classified into two main groups:

Group A (Control group): 3 groups (7 mice each)

A ₁: Non-infected non-treated mice (negative control).
A ₂: Toxoplasma-infected hypertensive non treated mice (positive control).
A ₃: Toxoplasma-infected diabetic non treated mice (positive control).

Group B (Basil-treated group) = 8 groups/7 mice each

B ₁: Hypertensive mice treated by basil before T. gondii infection (prophylactic group).
B ₂: Toxoplasma-infected hypertensive mice, treated by spiramycin (control drug).
B ₃: Toxoplasma-infected hypertensive mice treated by basil.
B ₄: Toxoplasma-infected hypertensive mice, treated by both basil and spiramycin.
B ₅: Diabetic mice treated by basil before T. gondii infection (prophylactic group).
B ₆: Toxoplasma-infected diabetic mice treated by spiramycin (control drug).
B ₇: Toxoplasma-infected diabetic mice, treated by basil.
B ₈: Toxoplasma-infected diabetic mice, treated by both basil and spiramycin.

4.1. Spiramycin drug:

Spiramycin 3 million international units (M.I.U) was used in a tablet form (Pharaonia Pharmaceuticals company, Egypt). Spiramycin tablets were smashed into powder, and phosphate buffered saline (PBS) of PH 7.2 was used in dissolving process. It was given at a dose of 200 mg/kg/day for 14 consecutive days (Grujić et al. 2005 ; Abdel Hamed et al. 2019). The drug was given orally to mice using esophageal tube either singly or in combination with basil to the chronic Toxoplasma infected hypertensive (B₂ and B₄) and diabetic (B₆ and B₈) mice.

4.2. Basil

Pure basil was used in oil form (El Captain Company, Cap pharm, Egypt).

Basil was used in oil form where it was dissolved in dimethyl sulfoxide (DMSO) solvent (Manjunath and Shivaprakash, 2013). Prepared solutions were stored in labeled containers with the drug name. Basil was given at a dose of 20 ml/kg /day for 14 consecutive days. The drug was given orally to mice using esophageal tube to chronic T. gondii infected hypertensive groups (B₃, B₄) and diabetic groups (B₇, B₈) mice. For prophylactic groups (B₁, B₅), the drug was given at a dose of 20 ml/kg /day for 14 consecutive days before Toxoplasma infection (de Almeida et al., 2007).

4. Induction of hypertension

Hypertension was induced by administration of oral, high salt solution of 8% NaCl to mice groups (B₁ to B₄) in a single dose per kg of animal weight given for 7 consecutive days. To check for induced hypertension, the blood pressure (BP) level was measured on experimental day 14 from a tail vein using tail cuff technique (LSI Lectica (LE5001) device). BP level was measured three times per week till end of study. Mice were considered hypertensive when systolic blood pressure (Bp) is more than 140 and diastolic Bp is higher than 90 (Van Vliet and Montani, 2008).

5. Induction of diabetes mellitus

T1DM was induced by oral administration of a solution of Alloxan monohydrate (ALDRICH chemistry, southern Africa) at 2% diluted in 0.9% saline to mice groups (B₅ - B₈). It was administered to mice in a single dose (40 mg/kg) for five consecutive days. Induced hyperglycemia was checked by measuring the blood glucose level on experimental day 14 from a tail vein blood sample using a glucometer (Fine test, Auto-coding premium, INFOPIA device). Blood glucose level was estimated 3 times per week till end of study. Blood glucose level more than 180 mg /dl represented diabetes (de Carvalho et al. 2003).

6. Induction of infection

6. Induction of Toxoplasma infection

6.1. T. gondii isolation, preparation of the inoculum and tissue cysts counting:

T. gondii ME49 chronic strain was obtained from TBRI. Brains of infected mice with T. gondii tissue cysts (6–8 weeks prior to the experiment) were isolated after mice sacrificing and used for induction of chronic infection in experimental mice.

Brain tissues were collected and homogenized. About 20 µl of the homogenized brain tissues were stained with Giemsa stain and examined by oil immersion lens (× 100). T. gondii tissue cysts were counted in 10 different fields, and the average number of tissue cysts in 20 µl was calculated (Elfadaly et al. 2015).

6.2. Mice infection and sacrificing:

Each mouse was infected by 200 µl of PBS containing 1000 ± 200 tissue cysts isolated from Toxoplasma-infected mice by oral inoculation.

Animal scarification was done after 45 days in control groups A (A₁, A₂, A₃), after 47 days in groups (B₅-B₈) and 60 days post infection (dpi) in groups (B₁-B₄). Mice received anesthetic– anticoagulant solution (500 mg/kg thiopental and 100 unit/ml heparin) by intraperitoneal injection (Liang et al. 2003). Brain, spleen and kidney were excised from each mouse and subjected to histopathological examination to assess the histopathological changes.

7. Assessment of Drug efficacy

7.1. Parasitological Assessment

Part of each brain tissue was homogenized, and dissolved in 100 µl of (PBS), PH 7.2. About 20 µl of this fluid were stained with Giemsa stain and microscopically examined. The tissue cysts were counted in brain sections using oil immersion lens (×100) to evaluate the reduction in their numbers. The mean number of tissue cysts in ten different fields of each mouse’s brain and the mean number of the 7 mice were calculated (Elfadaly et al. 2015).

7.2. Histopathological examination

To evaluate the pathological changes that occurred in the brain, kidney and spleen tissues, parts of these tissues were collected in formol-water 7%, dehydrated in ascending series of ethyl alcohol then cleared in xylene and embedded in paraffin.

Sections 5µ thick each were cut and stained with hematoxylin and eosin (H&E) stain (Drury & Wallington, 1980). All stained tissue sections were examined using light microscopy as well as digital slide scanner (Leica APERIO LV1 microscopy).

8. Statistical analysis of data:

Data were coded and entered and analyzed using the statistical package for the Social Sciences (SPSS) version 25 (IBM Corp., Armonk, NY, USA) for Microsoft Windows 10. Descriptive analysis of the results in the form of (minimum, maximum, median, and IQR: Interquartile Range) calculation for quantitative data. Statistical analysis was carried out using independent samples Kruskal Wallis test followed by pairwise comparison analysis. Differences were considered significant at p-values ≤ 0.05. Total p-value for Kruskal Wallis test was calculated and written, p-values of pairwise comparison analysis were written and expressed as small letters (^a,b,c,d,e).

Percent reduction (PR) of Toxoplasma gondii cysts; that expressed the decrease in cysts' number of treated group in comparison to that of infected untreated control group was calculated by the formula:

$$\:\text{P}\text{R}=\frac{(\text{C}\text{y}\text{s}\text{t}\text{s}\:\text{N}\text{o}\:\text{i}\text{n}\:\text{u}\text{n}\text{t}\text{r}\text{e}\text{a}\text{t}\text{e}\text{d}\:\text{c}\text{o}\text{n}\text{t}\text{r}\text{o}\text{l}\:\text{g}\text{r}\text{o}\text{u}\text{p}-\text{C}\text{y}\text{s}\text{t}\text{s}\:\text{N}\text{o}\:\text{i}\text{n}\:\text{t}\text{h}\text{e}\:\text{t}\text{r}\text{e}\text{a}\text{t}\text{e}\text{d}\:\text{g}\text{r}\text{o}\text{u}\text{p})}{\text{C}\text{y}\text{s}\text{t}\text{s}\:\text{N}\text{o}\:\text{i}\text{n}\:\text{u}\text{n}\text{t}\text{r}\text{e}\text{a}\text{t}\text{e}\text{d}\:\text{c}\text{o}\text{n}\text{t}\text{r}\text{o}\text{l}\:\text{g}\text{r}\text{o}\text{u}\text{p}}\:\times\:100$$

Results of parasitological examination:

Unstained brain tissue cysts appeared round to oval with thin cyst wall and their size ranged from 5–50 µm in diameter. Whereas Giemsa-stained tissue cysts were purple stained containing from a few to hundreds bradyzoites with terminal nucleus (Fig. 1).

Results of hypertensive groups:

In the hypertensive positive control group (A₂), the median number of Toxoplasma tissue cysts was 1600. The median tissue cyst count for basil prophylactic group (B1) was 1034 with 35.3% reduction rate in comparison to infected control group (P value ≤ 0.05). Infected hypertensive mice treated with spiramycin (B2), basil (B3), and combined basil and spiramycin therapy (B4) showed median tissue cyst counts of 930, 950 and 615 with reduction rates (41.4%, 42.1% and 60.5% respectively), with statistical difference in comparison to the infected positive control group (A2) (P value ≤ 0.05) (Table 1) (Fig. 2).

Table 1

Median count and % reduction of *Toxoplasma* brain tissue cysts in infected non-treated and basil-treated hypertensive mice groups.
Tissue cyst count	Median	Minimum	Maximum	IQR	% reduction	P
Positive control group (A₂)	1600	1440	2040	300.0	----	0.001*
Prophylactic group (B₁)	1034	942	1225	200.0	35.3%
Spiramycin treated group (B₂)	930 ^a	796	1220	260.0	41.4%
Basil treated group (B₃)	950 ^a	600	1200	270.0	42.1%
Combined therapy group (B₄)	615 ^a,b	480	850	271.0	60.5%
IQR: Interquartile range/ *P-value ≤ 0.05 was statistically significant. Statistical analysis was carried out using the Kruskal Wallis test followed by pairwise comparison analysis.
- ^a Significantly different from controls (group A₂) at P ≤ 0.05.
- ^b Significantly different from hypertensive mice (group C₁) at P ≤ 0.05.
- ^c Significantly different from hypertensive mice (group C₂) at P ≤ 0.05.

There was a decrease in blood pressure readings in the groups treated with basil (B₃, B₄) (136/85 and 135/83 respectively), with no statistically significant difference between the hypertensive mice treated with basil and the hypertensive control group regarding the level of systolic and diastolic blood pressure (P = 0.202 and 0.156 respectively) (Table 2).

Table 2

Blood pressure level in experimentally infected hypertensive non-treated and basil-treated hypertensive mice group.
Blood pressure level (mm Hg)	Median	Minimum	Maximum	IQR	P
Hypertensive control group (A₂)	140/90	135/85	142/95	5 /5	P1 = 0.202 P2 = 0.156
Prophylactic group (B₁)	138/88	120/80	145/95	10 /10
Spiramycin treated group (B₂)	140/90	135/85	142/90	6/5
Basil treated group (B₃)	136/85	132/80	140/90	13/5
Combined therapy group (B₄)	135/83	130/80	138 /89	9/10
IQR: Interquartile range/ P1 for systolic blood pressure/ P2 for diastolic blood pressure/ P-value ≤ 0.05 was statistically significant. Statistical analysis was carried out using the Kruskal Wallis test.

Results of diabetic groups:

In the diabetic control group (A₃), the median tissue cyst count was 1570. In the diabetic basil groups (B₅-B₈), the highest reduction in median tissue cyst count was found in infected diabetic mice treated with spiramycin (B₆) where their count was 725 with reduction rates (54%). This is followed by the group treated with combined basil and spiramycin therapy (B₈) and those treated with basil only (B₇) with reduction rate 46.6 and 43% respectively. The difference was statistically different in comparison to the infected control group (A₃). While the median number of T. gondii tissue cysts in the prophylactic group (B₅) was 1015 with 38.2% reduction which was statistically non different from control group (Table 3) (Fig. 3).

Table 3

Median count and % reduction of *Toxoplasma* brain tissue cysts in infected non-treated and basil-treated diabetic mice groups.
Tissue cyst count	Median	Minimum	Maximum	IQR	% reduction	P
Diabetic control group (A₃)	1570	1420	2025	270	----	0.001*
Prophylactic group (B₅)	1015	800	1200	895	38.2%
Spiramycin treated group (B₆)	725 ^a	600	1000	600	54%
Basil treated group (B₇)	873 ^a	753	1300	800	43%
Combined therapy group (B₈)	842 ^a	753	1023	800	46.6%
IQR: Interquartile range/ *P-value ≤ 0.05 was statistically significant. Statistical analysis was carried out using the Kruskal Wallis test followed by pairwise comparison analysis.
- ^a Significantly different from controls (group A₃) at P ≤ 0.05.
- ^b Significantly different from diabetic mice (group C₅) at P ≤ 0.05.
- ^c Significantly different from diabetic mice (group C₆) at P ≤ 0.05.

Random blood sugar level in basil treated groups (B₇ & B₈) was lower than diabetic control group (A₃) with non-statistically significant difference (P = 0.193) (Table 4).

Table 4

Random blood sugar level in experimentally infected diabetic non-treated mice and basil-treated diabetic mice group.
RBS (mg/dl)	Median	Minimum	Maximum	IQR	P
Control Group (A₃)	179	166	204	21	0.054
Prophylactic Group (B₅)	176	139	185	23
Spiramycin treated Group (B₆)	177	159	190	16
Basil treated Group (B₇)	166	158	179	16
Combined therapy Group (B₈)	169	155	181	21
IQR: Interquartile range/ P-value ≤ 0.05 was statistically significant. Statistical analysis was carried out using the Kruskal Wallis test.

Results of histopathological examination:

Histopathological parameters of brain sections:

Negative control group (A₁) showed normal brain tissue, normal six layers of cerebral cortex, normal neurons and neuroglial cells. The brain sections from infected control groups A₂ (hypertensive) and A₃ (diabetic) showed diffuse degeneration of neural cells, marked neutrophil vacuolation, dilated congested blood vessels with perivascular space, chronic inflammatory infiltrate associated with presence of tissue cysts of T. gondii (Fig. 4).

The brain sections from infected hypertensive groups treated by spiramycin (B₂) and basil (B₃) showed moderate degenerated neural cells, moderate congestion and neurophil vacuolation with minimal mononuclear cell infiltrate in absence of tissue cyst. Whereas the brain sections from infected hypertensive group treated by combination of basil and spiramycin drugs (B₄) showed scattered degenerated neural cells, moderate congestion and minimal neurophil vacuolation with mild mononuclear cell infiltrate (Fig. 5).

The brain sections from infected diabetic groups treated by spiramycin (B₆), basil (B₇) and the combination of basil and spiramycin drugs (B₈) showed scattered degenerated neural cells, moderate congestion and moderate neurophil vacuolation with mild mononuclear cell infiltrate (Fig. 6).

Histopathological parameters of the spleen sections:

Negative control group (A₁) showed normal splenic tissue consisting of white pulp formed of lymphoid follicles with central arterioles and red pulp containing splenic sinusoids. While spleen sections from infected control groups A₂ (hypertensive) and A₃ (diabetic) showed marked disruption of splenic architecture with increase in megakaryocytes and multiple T. gondii tissue cysts associated with foci of hemorrhage (Fig. 7).

The spleen sections from infected hypertensive group treated by spiramycin drug (B₂) showed moderate edema and foci of extravasation RBCs with absence of T. gondii tissue cysts. While spleen tissue from infected hypertensive group treated by basil drug (B₃) showed mild disruption of splenic architecture and few scattered T. gondii tissue cysts surrounded by megakaryocytes. Spleen section from infected hypertensive group treated by combination of basil and spiramycin drugs (B₄) showed slightly preserved normal splenic architecture with moderate edema and foci of extravasation RBCs in absence of T. gondii tissue cysts (Fig. 8).

Spleen section from infected diabetic group treated by spiramycin drug (B₆) showed moderate congestion and foci of hemorrhage in absence of T. gondii tissue cysts, while spleen section from infected diabetic group treated by basil drug (B₇) showed mild disruption of splenic architecture with foci of hemorrhage and congestion and presence of scattered T. gondii tissue cysts. Spleen section from infected diabetic group treated by combination of basil and spiramycin drugs (B₈) showed slightly preserved normal splenic architecture with moderate edema and foci of extravasation RBCs (Fig. 8).

Histopathological parameters of kidney:

Renal section from negative control group (A₁) showed normal renal tissue, normal glomeruli consisting of capillary tuft encircled by Bowman’s capsule, distal convoluted tubules with large lumen and proximal convoluted tubules with round lumen and eosinophilic cytoplasm. While renal section from infected control A₂ (hypertensive) and A₃ (diabetic) group showed marked capillaries glomerular tuft congestion with Bowman's capsule swelling and dilatation. The surrounding renal tubules showed diffuse severe acute tubular necrosis (Fig. 9).

Renal sections from infected hypertensive groups treated by spiramycin (B₂), basil drug (B₃) and combination of basil and spiramycin drugs (B₄) showed glomeruli congestion and distended of Bowman's capsule by fibrin which compressed the capillary tuft with moderate interstitial edema and moderate renal tubular injury consisted of degenerative hydropic changes and intraluminal epithelial cell cast (Fig. 10).

Renal sections from infected diabetic groups treated by spiramycin (B₆), basil drug (B₇) and combination of basil and spiramycin drugs (B₈) showed moderate capillaries glomerular tuft congestion with moderate renal tubular injury consisted of degenerative hydropic changes and intraluminal epithelial cell cast (Fig. 10).

Toxoplasmosis is one of the most important public health problems that is endemic worldwide and affects one third of the human population (Adem and Ame, 2023).

In immunocompetent patients, toxoplasmosis manifests as acute forms presenting with mild or asymptomatic disease, and chronic forms that establishes as latent tissue cysts. In immunocompromised patients, chronic toxoplasmosis may reactivate, leading to a potentially life-threatening condition (Almeria and Dubey, 2021).

Toxoplasmosis has also been linked to occurrence of several diseases including diabetes melliuts (DM) and hypertension (Laboudi, 2017). The probable correlation between toxoplasmosis and chronic diseases including DM and HTN is still understood.

Basil (O. basilicum) is a culinary herb of the family Lamiaceae (mints) which grows in tropical and sub-tropical climates. Basil is widely used as powerful flavoring agents, and for perfumery purposes in many countries (Mostafavi et al., 2019). It has variable pharmacological activities, such as analgesic, anti-inflammatory, and antiparasitic effect (Elazab et al., 2021 and Barreto-Brandão et al., 2022).

Basil has hypolipidemic and hypoglycemic effect through its action on α-amylase and α-glucosidase (Rodrigues et al., 2016 and Al-Snafi, 2021). Also, it has also anti-hypertensive effect through inhibiting angiotensin-converting enzyme (Chaudhary et al., 2016 and Al-Snafi, 2021).

In the present study, there was a reduction in Toxoplasma median tissue cyst counts of infected hypertensive mice treated with spiramycin (B₂) and basil (B₃) (41.4% and 42.1% respectively). The combined therapy (basil and spiramycin group, B₄) showed the highest reduction rate 60.5%. This can be explained by basil antiparasitic, antioxidant and anti-inflammatory activities (Ch et al., 2015 and Dhama et al., 2021).

However, the median tissue cyst counts for prophylactic group (B₁) showed the lowest reduction rate 35.3% in comparison to infected control group (A₂).

Elazab et al. (2021) studied the anti-parasitic effectiveness of basil oil against T. gondii. They demonstrated that basil oil inhibited the development and multiplication of T. gondii in vitro. The authors concluded that basil oil has the potential to act as a natural anti-parasitic agent against T. gondii. Also, Abdou et al. (2022) showed that T. gondii tachyzoites were significant inhibited by an ethanolic extract of basil with reduction rate of 46.6%. The authors elucidated this potent effect against T. gondii as a part of its antimicrobial properties and anti-oxidative effects.

Other previous studies showed that basil inhibits parasite multiplication and alters ultrastructure mostly in the nucleus in T. cruzi epimastigote and bloodstream trypomastigote forms. It also has anti-Leishmania, anti-malaria, anti-Giardia lamblia and anti-Trichomonas vaginalis effects (Eldin and Badawy, 2015; Ch et al., 2015; Dhama et al., 2021 and Al-Snafi, 2021).

There was a decrease in blood pressure readings in the groups treated with basil (B₃, B₄), with no statistically significant difference between the hypertensive mice treated with basil and the hypertensive control group regarding the level of systolic and diastolic blood pressure. This result can be clarified by that the tested dose isn't sufficient to reduce blood pressure and hypertension needs higher doses to be tested for longer duration.

This concept is confirmed by Rysz et al. (2017) who found that a daily dose of 30 mg/kg of basil is beneficial in lowering both systolic and diastolic blood pressure in humans. Moreover, the results of the study conducted by Ratta et al. (2021) showed that giving hypertensive patients 128 mg of basil leaves once daily resulted in a statistically significant reduction in the blood pressure (p = < 0.0001).

In fact, several mechanisms have been discussed to explain blood pressure-lowering effect of basil. Basil contains eugenol and rosmarinic acid which relax blood vessels and enhance blood flow, potentially aiding to hypertension control. Others discovered that eugenol inhibits calcium channels resulting in a significant drop in both systolic and diastolic blood pressure (Rysz et al., 2017; Ratta et al., 2021 and Amini et al., 2023).

To best of our knowledge this is the first study that assess the prophylactic and therapeutic effects of basil against chronic toxoplasmosis in hypertensive mice, and there are no research focusing entirely on basil for this goal.

Concerning diabetic groups treated with basil, the present study showed that the highest reduction in Toxoplasma median tissue cyst count was found in infected diabetic mice treated with spiramycin (B₆) followed by combined basil and spiramycin therapy (B₈) and those treated with basil only (B₇) (54%, 46.6 and 43% respectively). The difference was statistically different in comparison to the infected control group (A₃). While the median number of T. gondii tissue cysts in the prophylactic group (B₅) was 1015 with 38.2% reduction. Our finding suggests that basil may have an antiparasitic effect against T. gondii.

This agreed with the results of Widjaja and Savira (2019) who showed that, an ethanol extract of basil leaves can lower blood glucose in diabetic rats. However, there was no statistical significance between normal control group and group of diabetic rats treated with basil leaves. Several mechanisms were suggested for this effect. Basil may aid in blood sugar regulation by increasing insulin sensitivity, decreasing oxidative damage, degrading complex dietary carbohydrates to monosaccharides and reducing glucose absorption via inhibition of carbohydrate metabolizing enzymes and increasing hepatic glucose mobilization (Ademiluyi et al., 2016; Ezeani et al., 2017; Dhama et al., 2021 and Al-Snafi, 2021).

However, the precise mechanisms underlying basil's possible blood pressure-lowering actions and its possible benefits for diabetes treatment is still under research. More researches are needed, however, to precisely investigate basil's prophylactic and therapeutic effects on chronic toxoplasmosis in both in vitro and in vivo models.

In the present study, the histopathological examination of the brain from infected control groups (A₂ and A₃) showed multiple T. gondii tissue cysts, diffuse degeneration of neural cells and marked neurophil vacuolation with dilated congested blood vessels. These results are consensus with those previously reported on Toxoplasma infected control group compared to the normal control group (Etewa et al., 2018; El-Sayad et al., 2020; Etewa et al., 2021; Barakat et al., 2022 and Ode et al., 2022).

Spiramycin treated groups (infected hypertensive group B₂ and in infected diabetic group B₆), showed few scattered degenerated neural cells and congestion in the brain tissue associated with minimal neutrophil vacuolation. This result was similar to many earlier studies (Etewa et al., 2018; Omar et al., 2021; Allam et al., 2021 and Allam et al., 2022).

In basil treated infected hypertensive groups (B₁, B₃ and B₄) and infected diabetic groups (B₅, B₇ and B₈), the brain tissue showed scattered degenerated neural cells and moderate congestion associated with mild to moderate neurophil vacuolation. Our results partially coincide with a study on experimental trypanosomiasis treated with essential oil of basil and found that treatment with basil decreased lesions, chromatolysis of neurons and improved the brain edema with moderately congested blood vessels (Razin et al., 2023).

In the current study, the spleen section from infected control groups A₂ and A₃ were infiltrated by multiple T. gondii tissue cysts with foci of hemorrhage and marked disruption of splenic architecture with increased megakaryocytes. These results were in accordance with Pereira et al. (2019) and Yahia et al. (2022) who showed that spleen tissue sections from the Toxoplasma infected group showed loss of architecture, markedly dilated blood sinusoids with fibrinoid material and multiple T. gondii cysts.

In spiramycin treated infected hypertensive group B₂ and infected diabetic group B₆, the spleen tissue showed moderate congestion and foci of hemorrhage with absence of T. gondii tissue cysts. This result is consensus with Etewa et al. (2018) where the spleen tissues revealed moderate decrease in the pathological alterations in Toxoplasma infected mice that were administered spiramycin treatment in comparison to the infected control group. Moreover, our results coincide with a study conducted by Allam et al. (2022) who revealed reduction of meant Toxoplasma stages count as well as decreased inflammation, and congestion within tissue sections of the liver and spleen of all treated mice by spiramycin.

In basil treated infected hypertensive groups (B₁, B₃ and B₄) and infected diabetic groups (B₅, B₇ and B₈), there was mild to moderate congestion and foci of hemorrhage with scattered or absence of T. gondii tissue cysts in spleen sections. Similarly, a study conducted by Razin et al. (2023) on experimental trypanosomiasis found that treatment with basil revealed an improvement in histopathological changes in the spleen.

In the present study, regarding histopathological examination of the kidney, the infected control groups A₂ and A₃ showed marked capillaries glomerular tuft congestion with Bowman's capsule swelling and dilatation with the surrounding renal tubules showed diffuse severe acute tubular necrosis. These results were comparable with Al-Sabawi et al. (2018) and Barakat et al. (2022) who recorded that there were degenerative and necrotic changes in the endothelial cells of the proximal and distal convoluted tubules of the kidney with sloughing of endothelial cells of the tubes on kidney sections with presence of multiple T. gondii tissue cysts in the lumen of proximal convoluted tubules.

In spiramycin treated infected hypertensive group B₂ and infected diabetic group B₆, the renal tissue demonstrated moderate congested capillary glomerular tuft and few tubules showed degenerative hydropic changes with intraluminal epithelial cell cast.

Similarly, a study conducted by Ibrahim et al. (2022) showed that administration of spiramycin showed moderate lymphocytic inflammatory cellular infiltrate. Also, Elkholy et al. (2023) demonstrated that spiramycin treated mice revealed few degenerated renal tubules with no inflammatory cell infiltrates with improvement in congestion. Moreover, co-treatment of spiramycin and nitrofurantoin improved renal injury and reduced renal damage without inflammatory cell infiltration and less congested capillaries.

In the present study, the basil treated groups (infected hypertensive groups (B₁, B₃ and B₄) and infected diabetic groups (B₅, B₇ and B₈) showed moderate congested capillary glomerular tuft with moderate renal tubular injury consisting of degenerative hydropic changes and intraluminal epithelial cell cast. These results showed some similarity with Razin et al. (2023) who reported that renal tissue treated by basil showed more or less normal with slight congestion and necrosis of tubules on experimental trypanosomiasis.

Basil showed promising an antiparasitic effect on toxoplasmosis in hypertensive and diabetic mice. Additional studies are needed to investigate the hypoglycemic and blood pressure-lowering effects of basil and to conduct other studies on many cases to determine whether T. gondii increases the chance of developing hypertension or diabetes.

Conflict of interest

The authors declare that they have no conflict of interest.

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Evaluation of Anti-Toxoplasma activity of Basil (Ocimum basilicum) in Experimentally Infected Diabetic or Hypertensive Mice

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Abstract

Figures

1. Introduction

2. Materials and Methods

1. Experimental animals

2. Ethical considerations

3. Experimental design

4. Drugs preparation and dose adjustment

4.1. Spiramycin drug:

4.2. Basil

4. Induction of hypertension

5. Induction of diabetes mellitus

6. Induction of infection

6.1. T. gondii isolation, preparation of the inoculum and tissue cysts counting:

6.2. Mice infection and sacrificing:

7. Assessment of Drug efficacy

7.1. Parasitological Assessment

7.2. Histopathological examination

8. Statistical analysis of data:

5. Results

6. Discussion

7. Conclusion

Declarations

Conflict of interest

References

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