# Evaluation of Acute and Subacute Toxicities ofPsidium guajava Methanolic Bark Extract: A Botanical with In Vitro Antiproliferative Potential

Authors: Hermione T. Manekeng; Armelle T. Mbaveng; Samuel A. Ntyam Mendo; Armel-Joseph D. Agokeng; Victor Kuete
Journal: Evidence-Based Complementary and Alternative Medicine
 (2019)

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## Abstract

The methanol crude extract of the bark ofPsidium guajava (guava) previously displayed interesting cytotoxic effects on a panel of human cancer cell lines. In the present work, we plan to determine the toxicological effects of this guava botanical in Wistar rats. Acute oral toxicity of the extract was carried out by administration of a single dose of 5000 mg/kg body weight to female rats in 14 days. Subacute toxicity was conducted by oral administration of the extract at daily doses of 250 mg/kg, 500 mg/kg, and 1000 mg/kg body weight, respectively, while rats in the control group received no extract. After 28 days of treatment, animals were sacrificed for hematological and biochemical studies. In the acute toxicity study, no mortality or signs of toxicity were recorded; hence, the median lethal dose (LD50) of the Psidium guajava bark extract is greater than 5000 mg/kg body weight. For the subacute toxicity study, significant variations in body weight, relative weight of organs, and biochemical parameters were observed in the animals treated at different doses of the plant extract compared to control animals. Histopathological analyses showed minor liver inflammation in females treated at the highest dose (1000 mg/kg). These results suggest that intake of a single high dose of the Psidium guajava bark extract is nontoxic, but repeat administration could exhibit mild organ toxicity.


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## Body

## 1. Introduction

Because of its great diversity in medicinal plants, nature gives humanity the gift of an immense therapeutic wealth [1]. The consumption of natural substances to cure and prevent diseases is old and universal; it plays an important role in access to basic health care for populations [2]. Research on the therapeutic potential of plants has flown over the years with a wealth of scientifically proven information, showing the considerable power of plants in the treatment of a wide range of diseases [3]. All parts of the plant that is the subject of this work especially leaves are traditionally recognized in the treatment of diarrhea, dysentery, gastrointestinal problems, gastric pain, respiratory problems, obesity, and hypertension [4, 5].Psidium guajava, commonly known as guava, is a plant of the family Myrtaceae, native to South America, but to date widespread throughout the tropical zone especially where the climate is favorable [6]. This medium-sized plant, up to 10 m tall, is used as food, and all its parts have pharmacological properties [7]. It has a plethora of laboratory-proven biological activities such as antimicrobial [8], antidiarrheal [9], hepatoprotective [10], antioxidant [11], and anticancer [12]. This latter activity has already been the subject of numerous studies, as the work of Salib and Michael [13] demonstrated that the aqueous extract of P. guajava leaves inhibits the viability of cancer cell lines DU-145 in a dose-dependent manner, while its essential oil has antiproliferative activity four times more potent than that of vincristine, a reference anticancer drug on P388 lines. Similarly, the methanol extract of the peel of its fruit has promising in vitro activities against MCF-7 cells of human breast cancer [14]. The methanolic bark extract of P. guajava revealed considerable cytotoxic activity against CEM/ADR5000 cells with a 50% inhibitory concentration (IC50) of 1.29 μg/mL, against HCT 116 cells (p53+/+) with an IC50 of 18.63 μg/mL, and against several other sensitive and resistant cells with IC50 values up to 62.64 μg/mL (towards MDA-MB-231 cells) [12].However, plants in addition to their effectiveness must be free of harmful side effects for the consumer because the main criterion for the selection of medicinal plants is above all safety [1]. But the literature reveals that existing plant efficacy data are far more abundant than those for toxicity [15]. In this regard, the literature offers insufficient information on the toxicological effects related to the use of P. guajava extracts. It is therefore imperative to obtain more toxicological data from this plant in order to ensure its safety vis-à-vis humans and especially for the development of new drugs [16]. Our study aims to evaluate the acute and subacute toxicities of the methanol extract of the P. guajava bark in Wistar rats.


## 2. Subjects and Methods

### 2.1. Plant Material

The bark ofP. guajava used in this work was harvested in West Cameroon (in the locality of Dschang) in February 2018. A sample of this plant was authenticated by Mr. Jumbam Blaise at the National Herbarium of Cameroon (HNC) in Yaoundé compared to the reference sample number 2884/SRF/Cam.


### 2.2. Preparation of the Plant Extract

The plant was cleaned and dried under the sun for a week and ground. The powder obtained was macerated in methanol for 48 hours in the proportion of 1/3. The mixture was then filtered through Whatman No. 1 paper and concentrated using a rotary evaporator (Buchi R-200) at 65°C. The extract obtained was dried in an oven at 40°C to completely evaporate the residual solvent. After complete drying, the extract was weighed and stored in a refrigerator at 4°C.


### 2.3. Experimental Animals

Three nulliparous and nonpregnant female rats of age 12 weeks were used for the acute toxicity study. For the subacute toxicity study, 32 animals weighing between 170 and 245 g (16 males and 16 females) including nulliparous and nonpregnant females were used. All these animals were raised under experimental conditions at the animal house of Department of Biochemistry, University of Dschang. They were kept at room temperature, and the lighting sequence was that offered by the day and night cycles. The animals were individualized one week before the start of the experiment; they ate a standard food [17] and received drinking water ad libitum.


### 2.4. Acute Oral Toxicity: Experimental Procedure

The acute toxicity test was conducted according to OECD Test Guideline 425 [18] for acute oral toxicity. Animals were fasted for 12 hours and weighed before undergoing intragastric feeding. They received the P. guajava extract at a single dose of 5000 mg/kg b.w. After administration, the animals were deprived again of food for 4 hours during which they were observed individually to note any manifestation of toxicity (reduced mobility, aggressiveness, faeces appearance, and sensitivity to noise and pain). They were then observed daily for 14 days for signs of morbidity or mortality. On the 15th day of the test, animals were anesthetized with chloroform vapor and sacrificed for macroscopic observation of organs (liver, kidney, spleen, heart, and lungs).


### 2.5. Subacute Toxicity

This test was conducted according to OECD Guideline 407 [19]. In brief, 32 rats were randomly divided into 4 groups of 08 animals each (04 males and 04 females). Group 1 rats were used as controls and received daily distilled water as a vehicle, while rats in groups 2, 3, and 4 received the P. guajava extract at doses of 250, 500, and 1000 mg/kg b.w., respectively, by intragastric gavage each day for 28 days. During this period, the animals were examined daily for signs of toxicity; their mass and their food consumption were recorded every 02 and 03 days, respectively.


### 2.6. Sacrifice of Animals and Collection of Blood, Urine, and Organs

On the 29th day, after a fast of 12 h, all animals were stressed in order to collect the urine; they were subsequently anesthetized with chloroform vapor, and the blood samples were taken by cardiac puncture in EDTA tubes and without EDTA. The first samples were for the determination of hematological parameters and the second for the analysis of biochemical parameters. The organs (kidney, liver, heart, lung, and spleen) were removed, cleaned with physiological saline, and weighed to calculate their relative weights. Liver and kidney samples from each dose group were preserved in 10% formalin solution for histopathological studies.


### 2.7. Determination of Weight Gain and Relative Weight of Organs

The animal’s body weight determined before and at the end of the test made it possible to calculate the weight gain by subtracting the starting body weight from that at the end of the test. Relative weight of organs was calculated using the following formula:(1)Relative weight=weight of the organanimal body weight×100.


### 2.8. Hematological Parameters

Plasma components such as white blood cells, monocytes, granulocytes, red blood cells, hemoglobin, hematocrit, mean corpuscular volume, mean corpuscular hemoglobin, lymphocytes, and platelets were listed using an automated analyzer hematology.


### 2.9. Biochemical Parameters

Serum activities of AST and ALT, as well as serum levels of total protein, total cholesterol, HDL cholesterol, creatinine, and urea, were determined using specific commercial kits (SGM Italia).


### 2.10. Histopathological Analysis

Liver and kidney samples were stored in 10% formalin and processed by conventional techniques. These tissues were subsequently dehydrated in increasing concentration of alcohol, inserted into paraffin, and cut into sections of 4-5μm. These paraffin sections (5 μm thick) were stained with hematoxylin-eosin prior to microscopic examination [20].


### 2.11. Statistical Analysis

Results obtained were presented as average ± standard deviation; GraphPad Prism software version 5 was used for one-way analysis of variance followed by the Newman–Keuls multiple comparison test. Values were considered significant at the 5% probability level (p<0.05).


## 2.1. Plant Material

The bark ofP. guajava used in this work was harvested in West Cameroon (in the locality of Dschang) in February 2018. A sample of this plant was authenticated by Mr. Jumbam Blaise at the National Herbarium of Cameroon (HNC) in Yaoundé compared to the reference sample number 2884/SRF/Cam.


## 2.2. Preparation of the Plant Extract

The plant was cleaned and dried under the sun for a week and ground. The powder obtained was macerated in methanol for 48 hours in the proportion of 1/3. The mixture was then filtered through Whatman No. 1 paper and concentrated using a rotary evaporator (Buchi R-200) at 65°C. The extract obtained was dried in an oven at 40°C to completely evaporate the residual solvent. After complete drying, the extract was weighed and stored in a refrigerator at 4°C.


## 2.3. Experimental Animals

Three nulliparous and nonpregnant female rats of age 12 weeks were used for the acute toxicity study. For the subacute toxicity study, 32 animals weighing between 170 and 245 g (16 males and 16 females) including nulliparous and nonpregnant females were used. All these animals were raised under experimental conditions at the animal house of Department of Biochemistry, University of Dschang. They were kept at room temperature, and the lighting sequence was that offered by the day and night cycles. The animals were individualized one week before the start of the experiment; they ate a standard food [17] and received drinking water ad libitum.


## 2.4. Acute Oral Toxicity: Experimental Procedure

The acute toxicity test was conducted according to OECD Test Guideline 425 [18] for acute oral toxicity. Animals were fasted for 12 hours and weighed before undergoing intragastric feeding. They received the P. guajava extract at a single dose of 5000 mg/kg b.w. After administration, the animals were deprived again of food for 4 hours during which they were observed individually to note any manifestation of toxicity (reduced mobility, aggressiveness, faeces appearance, and sensitivity to noise and pain). They were then observed daily for 14 days for signs of morbidity or mortality. On the 15th day of the test, animals were anesthetized with chloroform vapor and sacrificed for macroscopic observation of organs (liver, kidney, spleen, heart, and lungs).


## 2.5. Subacute Toxicity

This test was conducted according to OECD Guideline 407 [19]. In brief, 32 rats were randomly divided into 4 groups of 08 animals each (04 males and 04 females). Group 1 rats were used as controls and received daily distilled water as a vehicle, while rats in groups 2, 3, and 4 received the P. guajava extract at doses of 250, 500, and 1000 mg/kg b.w., respectively, by intragastric gavage each day for 28 days. During this period, the animals were examined daily for signs of toxicity; their mass and their food consumption were recorded every 02 and 03 days, respectively.


## 2.6. Sacrifice of Animals and Collection of Blood, Urine, and Organs

On the 29th day, after a fast of 12 h, all animals were stressed in order to collect the urine; they were subsequently anesthetized with chloroform vapor, and the blood samples were taken by cardiac puncture in EDTA tubes and without EDTA. The first samples were for the determination of hematological parameters and the second for the analysis of biochemical parameters. The organs (kidney, liver, heart, lung, and spleen) were removed, cleaned with physiological saline, and weighed to calculate their relative weights. Liver and kidney samples from each dose group were preserved in 10% formalin solution for histopathological studies.


## 2.7. Determination of Weight Gain and Relative Weight of Organs

The animal’s body weight determined before and at the end of the test made it possible to calculate the weight gain by subtracting the starting body weight from that at the end of the test. Relative weight of organs was calculated using the following formula:(1)Relative weight=weight of the organanimal body weight×100.


## 2.8. Hematological Parameters

Plasma components such as white blood cells, monocytes, granulocytes, red blood cells, hemoglobin, hematocrit, mean corpuscular volume, mean corpuscular hemoglobin, lymphocytes, and platelets were listed using an automated analyzer hematology.


## 2.9. Biochemical Parameters

Serum activities of AST and ALT, as well as serum levels of total protein, total cholesterol, HDL cholesterol, creatinine, and urea, were determined using specific commercial kits (SGM Italia).


## 2.10. Histopathological Analysis

Liver and kidney samples were stored in 10% formalin and processed by conventional techniques. These tissues were subsequently dehydrated in increasing concentration of alcohol, inserted into paraffin, and cut into sections of 4-5μm. These paraffin sections (5 μm thick) were stained with hematoxylin-eosin prior to microscopic examination [20].


## 2.11. Statistical Analysis

Results obtained were presented as average ± standard deviation; GraphPad Prism software version 5 was used for one-way analysis of variance followed by the Newman–Keuls multiple comparison test. Values were considered significant at the 5% probability level (p<0.05).


## 3. Results

### 3.1. Acute Oral Toxicity

In this study, no death was recorded following the administration of 5000 mg/kg b.w. of theP. guajava methanolic bark extract. The animals were apparently healthy and showed no signs of toxicity at this dose extract. Macroscopic observation of organs (liver, kidney, heart, spleen, and lung) showed no alteration. Thus, the LD50 was greater than 5000 mg/kg b.w. Animals’ body weight during the test is recorded in Table 1.Table 1
Body weight of animals during acute toxicity.
Animals
Initial weight (g)
Final weight (g)
Weight gain (g)
01
197
198
1
02
188
190
2
03
189
190
1


### 3.2. Subacute Oral Toxicity

#### 3.2.1. Weight Growth and Food Intake

Body weight changes in females treated at different doses of the extract during the four weeks of the study presented an almost constant body weight compared to those in the control whose evolution was growing (Figure1). On the contrary, in male rats treated with the P. guajava methanolic bark extract, body weight slightly increased (Figure 2). All female and male animals that received the plant extract had a significantly lower (p<0.05) food intake (Figures 3 and 4).Figure 1
Effect of oral administration of thePsidium guajava extract on body weight changes of female rats during 28 days of the subacute toxicity study.Figure 2
Effect of oral administration of thePsidium guajava extract on body weight changes of male rats during 28 days of the subacute toxicity study.Figure 3
Effect of oral administration of thePsidium guajava extract on food intake of female rats during 28 days of the subacute toxicity study. ∗indicates significant values at p<0.05.Figure 4
Effect of oral administration of thePsidium guajava extract on food intake of male rats during 28 days of the subacute toxicity study. ∗indicates significant values at p<0.05.


#### 3.2.2. Relative Weight of Organs

Relative weight values of organs of all animals are presented in Table2. In females, the liver and spleen decreased at all extract doses (p<0.001), while the kidney, heart, and lungs significantly increased (p<0.001) in weight compared to those in control rats. In males, decreased weight of the liver, spleen, and lung (all doses, p<0.001) and heart (500 and 1000 mg/kg, p<0.001) of animals treated with the plant extract has been observed; relative weight of the kidney showed no significant difference from that of the control.Table 2
Effect of oral administration of thePsidium guajava extract on the relative weight of organs and weight gain.
Parameters
Females
Males
Control
250 mg/kg
500 mg/kg
1000 mg/kg
Control
250 mg/kg
500 mg/kg
1000 mg/kg
Liver
3.364 ± 0.077c
2.928 ± 0.026b
2.962 ± 0.037b
2.842 ± 0.001a
3.276 ± 0.133c
2.770 ± 0.049b
2.491 ± 0.002a
2.694 ± 0.057b
Kidney
0.573 ± 0.007a
0.612 ± 0.001b
0.610 ± 0.005b
0.662 ± 0.0005c
0.632 ± 0.015a
0.633 ± 0.002a
0.633 ± 0.001a
0.630 ± 0.007a
Heart
0.301 ± 0.004a
0.362 ± 0.0006c
0.355 ± 0.002c
0.326 ± 0.007b
0.324 ± 0.002c
0.320 ± 0.006c
0.271 ± 0.006a
0.285 ± 0.002b
Lung
0.534 ± 0.002a
0.666 ± 0.005b
0.710 ± 0.003d
0.700 ± 0.005c
0.725 ± 0.010d
0.593 ± 0.022b
0.546 ± 0.003a
0.637 ± 0.0009c
Spleen
0.472 ± 0.006d
0.341 ± 0.007a
0.370 ± 0.017b
0.404 ± 0.005c
0.509 ± 0.038c
0.463 ± 0.006b
0.397 ± 0.005a
0.374 ± 0.001a
Weight gain (g)
65.2
−2.9
1.5
−2.7
60.1
12.5
26.5
19.2
Data are expressed as mean ± standard deviation.n = 8 (4/sex); the values in the same line for a given sex followed by different letters in exponent are significantly different (p<0.05) according to the Newman–Keuls multiple comparison test.


#### 3.2.3. Hematological Parameters

Administration of theP. guajava methanolic bark extract to animals increases (p<0.05) the red blood cell number and reduces the amount of platelets in females (1000 mg/kg). In males (1000 mg/kg), it resulted in an increase (p<0.01) in the platelet number. However, the other parameters showed no significant difference (p>0.05) at all tested doses compared to those in control animals. All of these data are presented in Table 3.Table 3
Effect of oral administration of thePsidium guajava extract on hematological parameters.
Parameters
Females
Males
Control
250 mg/kg
500 mg/kg
1000 mg/kg
Control
250 mg/kg
500 mg/kg
1000 mg/kg
WBC (103/μL)
7.05 ± 1.17a
9.16 ± 1.63a
6.36 ± 1.38a
10.05 ± 2.98a
5.30 ± 2.21a
6.40 ± 1.25a
9.46 ± 0.93a
7.95 ± 2.67a
Lymphocytes (%)
41.58 ± 3.03a
34.25 ± 5.85a
36.23 ± 0.97a
36.85 ± 2.52a
34.40 ± 5.65a
37.42 ± 3.27a
36.17 ± 3.87a
38.72 ± 1.53a
Monocytes (%)
7.63 ± 1.92a
7.76 ± 1.65a
7.13 ± 0.30a
11.30 ± 2.43a
8.36 ± 1.57a
6.36 ± 0.76a
8.20 ± 3.47a
9.23 ± 3.75a
Granulocytes (%)
9.20 ± 4.18a
23.73 ± 10.08a
20.40 ± 2.21a
15.00 ± 3.00a
23.10 ± 9.80a
18.80 ± 6.90a
19.47 ± 4.32a
13.33 ± 1.90a
PLT (103/μL)
677.3 ± 30.07b
735.7 ± 16.50b
751.0 ± 9.539b
494.0 ± 76.08a
396.0 ± 10.00a
438.0 ± 47.84a
529.7 ± 30.09a
691.0 ± 108.5b
MPV (fL)
11.83 ± 1.12a
10.87 ± 1.79a
10.93 ± 2.12a
9.46 ± 0.75a
9.83 ± 1.55a
9.13 ± 0.37a
8.80 ± 0.70a
11.00 ± 1.40a
RBC (106/μL)
7.29 ± 0.20a
7.32 ± 0.45a
7.05 ± 0.09a
8.08 ± 0.24b
6.34 ± 1.93a
8.07 ± 0.30a
7.89 ± 0.52a
7.67 ± 0.24a
HGB (g/dL)
16.20 ± 0.10a
16.40 ± 0.90a
15.85 ± 0.05a
16.10 ± 0.40a
16.25 ± 0.05a
17.07 ± 0.61a
17.05 ± 0.15a
17.00 ± 0.20a
HCT (%)
43.50 ± 1.00a
44.45 ± 2.45a
41.50 ± 1.31a
45.20 ± 2.10a
43.25 ± 0.95a
47.03 ± 0.80a
45.53 ± 1.88a
45.07 ± 4.53a
MCV (fL)
62.60 ± 3.98a
57.73 ± 2.80a
58.93 ± 1.06a
55.90 ± 3.89a
58.00 ± 0.50a
58.37 ± 1.41a
60.15 ± 0.55a
58.67 ± 4.10a
MCH (pg)
22.70 ± 0.88a
21.50 ± 0.50a
21.87 ± 0.66a
20.33 ± 1.42a
21.75 ± 0.35a
21.13 ± 1.29a
21.20 ± 0.81a
21.53 ± 0.80a
MCHC (g/dL)
37.20 ± 1.10a
37.40 ± 0.95a
37.37 ± 0.66a
36.43 ± 1.65a
37.55 ± 0.95a
36.27 ± 1.80a
36.63 ± 1.01a
36.90 ± 3.57a
WBC: white blood cells; PLT: platelets; MPV: mean platelet volume; RBC: red blood cells; HGB: hemoglobin; HCT: hematocrit; MCV: mean corpuscular volume; MCH: mean corpuscular hemoglobin; MCHC: mean corpuscular hemoglobin concentration. Data are expressed as mean ± standard deviation.n = 6 (3/sex); the values in the same line for a given sex followed by different letters in exponent are significantly different (p<0.05) according to the Newman–Keuls multiple comparison test.


#### 3.2.4. Urinary Biochemical Parameters

Values of parameters evaluated in the urine of animals are summarized in Table4. In females, there were a decrease in the creatinine level (all doses, p<0.05) and an increase in protein levels (500 and 1000 mg/kg, p<0.001). However, there was no significant difference (p>0.05) for urea between the treated and control females. In males, no significant difference (p>0.05) was observed for creatinine, while urea (p<0.001) and protein (p<0.01) increased at the doses 500 and 1000 mg/kg b.w.Table 4
Effect of oral administration of thePsidium guajava extract on urinary biochemical parameters.
Parameters
Females
Males
Control
250 mg/kg
500 mg/kg
1000 mg/kg
Control
250 mg/kg
500 mg/kg
1000 mg/kg
Creatinine (mg/dL)
53.07 ± 0.832c
42.00 ± 0.400a
38.80 ± 3.555a
48.53 ± 1.007b
64.13 ± 3.449a
63.87 ± 1.890a
63.87 ± 2.663a
58.40 ± 6.053a
Urea (mg/dL)
1921 ± 184.2ab
1618 ± 197.4a
1877 ± 84.59ab
2066 ± 118.4b
1579 ± 105.3a
1482 ± 99.63a
4355 ± 171.1c
2649 ± 30.39b
Proteins (g/dL)
0.2721 ± 0.042a
0.1837 ± 0.020a
0.7211 ± 0.135b
1.020 ± 0.040c
1.136 ± 0.112a
1.252 ± 0.023ab
1.306 ± 0.035b
1.395 ± 0.042b
Data are expressed as mean ± standard deviation.n = 8 (4/sex); the values in the same line for a given sex followed by different letters in exponent are significantly different (p<0.05) according to the Newman–Keuls multiple comparison test.


#### 3.2.5. Serum Biochemical Parameters

Investigations made on the animal’s sera made it possible to obtain data in Table5. In females, there was a decrease in HDL cholesterol levels (500 and 1000 mg/kg, p<0.05), triglycerides (p<0.01), total protein (p<0.001), and creatinine (p<0.05) at all doses. AST levels (p<0.01), total cholesterol (p<0.001), LDL cholesterol (p<0.01), and urea (p<0.01) increased for all doses of the extract compared to those in control animals, while ALT levels showed no significant difference. In males, there was no significant difference between triglyceride levels of the test animals and control animals (p>0.05). Total protein levels (500 and 1000 mg/kg, p<0.001), ALT (p<0.01), AST, total cholesterol, and LDL cholesterol (p<0.001) decreased significantly at all doses, while urea (1000 mg/kg, p<0.01), HDL cholesterol (p<0.01), and creatinine (p<0.001) increased for all extract doses.Table 5
Effect of oral administration of thePsidium guajava extract on serum biochemical parameters.
Parameters
Females
Males
Control
250 mg/kg
500 mg/kg
1000 mg/kg
Control
250 mg/kg
500 mg/kg
1000 mg/kg
ALT (U/I)
66.20 ± 1.968a
64.75 ± 2.404a
70.36 ± 5.099a
68.33 ± 1.758a
89.57 ± 2.362c
74.21 ± 3.148b
42.49 ± 5.613a
41.47 ± 3.573a
AST (U/I)
46.27 ± 2.310a
71.88 ± 1.817c
64.02 ± 2.667b
58.46 ± 5.535b
79.44 ± 0.873c
66.06 ± 2.197a
69.84 ± 1.155b
65.85 ± 1.671a
Total cholesterol (mg/dL)
47.14 ± 2.277a
62.99 ± 2.364c
61.27 ± 0.735c
52.70 ± 1.767b
90.69 ± 2.414d
64.79 ± 4.614b
53.19 ± 1.068a
70.51 ± 1.258c
HDL cholesterol (mg/dL)
35.72 ± 0.705b
34.88 ± 5.009b
26.57 ± 1.301a
21.04 ± 3.239a
31.74 ± 2.260a
42.24 ± 4.495b
49.80 ± 4.061c
56.92 ± 1.596d
LDL cholesterol (mg/dL)
22.25 ± 2.873a
38.32 ± 7.160b
44.58 ± 0.7714b
40.99 ± 4.274b
69.70 ± 0.777d
32.84 ± 6.584c
14.16 ± 4.669a
24.42 ± 0.809b
Triglycerides (mg/dL)
54.14 ± 0.885c
51.02 ± 1.113b
49.37 ± 0.692b
46.71 ± 1.113a
53.77 ± 0.692a
51.48 ± 1.262a
53.87 ± 1.301a
54.14 ± 1.113a
Creatinine (mg/dL)
1.036 ± 0.045c
0.571 ± 0.054b
0.631 ± 0.045b
0.488 ± 0.023a
0.4762 ± 0.038a
0.7738 ± 0.045b
0.8929 ± 0.071c
1.048 ± 0.086d
Urea (mg/dL)
69.91 ± 4.243a
82.41 ± 6.263b
90.28 ± 1.389b
84.26 ± 1.604b
70.83 ± 1.389a
74.07 ± 5.782a
78.70 ± 2.122a
89.81 ± 5.613b
Total proteins (g/dL)
4.926 ± 0.071c
4.192 ± 0.112b
4.072 ± 0.124b
3.501 ± 0.023a
3.426 ± 0.141b
3.401 ± 0.117b
3.015 ± 0.069a
2.878 ± 0.118a
ALT: alanine aminotransferase; AST: aspartate aminotransferase. Data are expressed as mean ± standard deviation.n = 8 (4/sex); the values in the same line for a given sex followed by different letters in exponent are significantly different (p<0.05) according to the Newman–Keuls multiple comparison test.


#### 3.2.6. Histological Studies

Histopathological examinations were performed on the kidney and liver of animals to verify whether they were damaged by the daily administration of theP. guajava methanolic bark extract; different observations are illustrated. In females (Figure 5), the liver appeared normal at doses 250 and 500 mg/kg b.w., but at the dose 1000 mg/kg, there was inflammation (infiltration of leucocytes). The kidney, on the contrary, showed no alteration although color changes were observed. However, in male rats (Figure 6), the liver as well as the kidney showed no damage at all dose levels tested.Figure 5
Liver (L) and kidney (K) sections of female rats showing effects of oral administration of thePsidium guajava extract over 28 days; L0, K0: control group; L1, K1: 250 mg/kg; L2, K2: 500 mg/kg; L3, K3: 1000 mg/kg. Indicators: A, branch of the hepatic portal vein; B, hepatocytes; c, leukocyte infiltration inflammation; s, sinusoid; v, centrolobular vein; G, glomerulus; EU, urinary space.
(a)
(b)
(c)
(d)
(e)
(f)
(g)
(h)Figure 6
Liver (L) and kidney (K) sections of male rats showing effects of oral administration of thePsidium guajava extract over 28 days; L0, K0: control group; L1, K1: 250 mg/kg; L2, K2: 500 mg/kg; L3, K3: 1000 mg/kg. Indicators: A, branch of the hepatic portal vein; B, hepatocytes; s, sinusoid; v, centrolobular vein; G, glomerulus; EU, urinary space; Ep, hepatic portal space; Cb, bile duct; P, proximal convoluted tubule.
(a)
(b)
(c)
(d)
(e)
(f)
(g)
(h)


## 3.1. Acute Oral Toxicity

In this study, no death was recorded following the administration of 5000 mg/kg b.w. of theP. guajava methanolic bark extract. The animals were apparently healthy and showed no signs of toxicity at this dose extract. Macroscopic observation of organs (liver, kidney, heart, spleen, and lung) showed no alteration. Thus, the LD50 was greater than 5000 mg/kg b.w. Animals’ body weight during the test is recorded in Table 1.Table 1
Body weight of animals during acute toxicity.
Animals
Initial weight (g)
Final weight (g)
Weight gain (g)
01
197
198
1
02
188
190
2
03
189
190
1


## 3.2. Subacute Oral Toxicity

### 3.2.1. Weight Growth and Food Intake

Body weight changes in females treated at different doses of the extract during the four weeks of the study presented an almost constant body weight compared to those in the control whose evolution was growing (Figure1). On the contrary, in male rats treated with the P. guajava methanolic bark extract, body weight slightly increased (Figure 2). All female and male animals that received the plant extract had a significantly lower (p<0.05) food intake (Figures 3 and 4).Figure 1
Effect of oral administration of thePsidium guajava extract on body weight changes of female rats during 28 days of the subacute toxicity study.Figure 2
Effect of oral administration of thePsidium guajava extract on body weight changes of male rats during 28 days of the subacute toxicity study.Figure 3
Effect of oral administration of thePsidium guajava extract on food intake of female rats during 28 days of the subacute toxicity study. ∗indicates significant values at p<0.05.Figure 4
Effect of oral administration of thePsidium guajava extract on food intake of male rats during 28 days of the subacute toxicity study. ∗indicates significant values at p<0.05.


### 3.2.2. Relative Weight of Organs

Relative weight values of organs of all animals are presented in Table2. In females, the liver and spleen decreased at all extract doses (p<0.001), while the kidney, heart, and lungs significantly increased (p<0.001) in weight compared to those in control rats. In males, decreased weight of the liver, spleen, and lung (all doses, p<0.001) and heart (500 and 1000 mg/kg, p<0.001) of animals treated with the plant extract has been observed; relative weight of the kidney showed no significant difference from that of the control.Table 2
Effect of oral administration of thePsidium guajava extract on the relative weight of organs and weight gain.
Parameters
Females
Males
Control
250 mg/kg
500 mg/kg
1000 mg/kg
Control
250 mg/kg
500 mg/kg
1000 mg/kg
Liver
3.364 ± 0.077c
2.928 ± 0.026b
2.962 ± 0.037b
2.842 ± 0.001a
3.276 ± 0.133c
2.770 ± 0.049b
2.491 ± 0.002a
2.694 ± 0.057b
Kidney
0.573 ± 0.007a
0.612 ± 0.001b
0.610 ± 0.005b
0.662 ± 0.0005c
0.632 ± 0.015a
0.633 ± 0.002a
0.633 ± 0.001a
0.630 ± 0.007a
Heart
0.301 ± 0.004a
0.362 ± 0.0006c
0.355 ± 0.002c
0.326 ± 0.007b
0.324 ± 0.002c
0.320 ± 0.006c
0.271 ± 0.006a
0.285 ± 0.002b
Lung
0.534 ± 0.002a
0.666 ± 0.005b
0.710 ± 0.003d
0.700 ± 0.005c
0.725 ± 0.010d
0.593 ± 0.022b
0.546 ± 0.003a
0.637 ± 0.0009c
Spleen
0.472 ± 0.006d
0.341 ± 0.007a
0.370 ± 0.017b
0.404 ± 0.005c
0.509 ± 0.038c
0.463 ± 0.006b
0.397 ± 0.005a
0.374 ± 0.001a
Weight gain (g)
65.2
−2.9
1.5
−2.7
60.1
12.5
26.5
19.2
Data are expressed as mean ± standard deviation.n = 8 (4/sex); the values in the same line for a given sex followed by different letters in exponent are significantly different (p<0.05) according to the Newman–Keuls multiple comparison test.


### 3.2.3. Hematological Parameters

Administration of theP. guajava methanolic bark extract to animals increases (p<0.05) the red blood cell number and reduces the amount of platelets in females (1000 mg/kg). In males (1000 mg/kg), it resulted in an increase (p<0.01) in the platelet number. However, the other parameters showed no significant difference (p>0.05) at all tested doses compared to those in control animals. All of these data are presented in Table 3.Table 3
Effect of oral administration of thePsidium guajava extract on hematological parameters.
Parameters
Females
Males
Control
250 mg/kg
500 mg/kg
1000 mg/kg
Control
250 mg/kg
500 mg/kg
1000 mg/kg
WBC (103/μL)
7.05 ± 1.17a
9.16 ± 1.63a
6.36 ± 1.38a
10.05 ± 2.98a
5.30 ± 2.21a
6.40 ± 1.25a
9.46 ± 0.93a
7.95 ± 2.67a
Lymphocytes (%)
41.58 ± 3.03a
34.25 ± 5.85a
36.23 ± 0.97a
36.85 ± 2.52a
34.40 ± 5.65a
37.42 ± 3.27a
36.17 ± 3.87a
38.72 ± 1.53a
Monocytes (%)
7.63 ± 1.92a
7.76 ± 1.65a
7.13 ± 0.30a
11.30 ± 2.43a
8.36 ± 1.57a
6.36 ± 0.76a
8.20 ± 3.47a
9.23 ± 3.75a
Granulocytes (%)
9.20 ± 4.18a
23.73 ± 10.08a
20.40 ± 2.21a
15.00 ± 3.00a
23.10 ± 9.80a
18.80 ± 6.90a
19.47 ± 4.32a
13.33 ± 1.90a
PLT (103/μL)
677.3 ± 30.07b
735.7 ± 16.50b
751.0 ± 9.539b
494.0 ± 76.08a
396.0 ± 10.00a
438.0 ± 47.84a
529.7 ± 30.09a
691.0 ± 108.5b
MPV (fL)
11.83 ± 1.12a
10.87 ± 1.79a
10.93 ± 2.12a
9.46 ± 0.75a
9.83 ± 1.55a
9.13 ± 0.37a
8.80 ± 0.70a
11.00 ± 1.40a
RBC (106/μL)
7.29 ± 0.20a
7.32 ± 0.45a
7.05 ± 0.09a
8.08 ± 0.24b
6.34 ± 1.93a
8.07 ± 0.30a
7.89 ± 0.52a
7.67 ± 0.24a
HGB (g/dL)
16.20 ± 0.10a
16.40 ± 0.90a
15.85 ± 0.05a
16.10 ± 0.40a
16.25 ± 0.05a
17.07 ± 0.61a
17.05 ± 0.15a
17.00 ± 0.20a
HCT (%)
43.50 ± 1.00a
44.45 ± 2.45a
41.50 ± 1.31a
45.20 ± 2.10a
43.25 ± 0.95a
47.03 ± 0.80a
45.53 ± 1.88a
45.07 ± 4.53a
MCV (fL)
62.60 ± 3.98a
57.73 ± 2.80a
58.93 ± 1.06a
55.90 ± 3.89a
58.00 ± 0.50a
58.37 ± 1.41a
60.15 ± 0.55a
58.67 ± 4.10a
MCH (pg)
22.70 ± 0.88a
21.50 ± 0.50a
21.87 ± 0.66a
20.33 ± 1.42a
21.75 ± 0.35a
21.13 ± 1.29a
21.20 ± 0.81a
21.53 ± 0.80a
MCHC (g/dL)
37.20 ± 1.10a
37.40 ± 0.95a
37.37 ± 0.66a
36.43 ± 1.65a
37.55 ± 0.95a
36.27 ± 1.80a
36.63 ± 1.01a
36.90 ± 3.57a
WBC: white blood cells; PLT: platelets; MPV: mean platelet volume; RBC: red blood cells; HGB: hemoglobin; HCT: hematocrit; MCV: mean corpuscular volume; MCH: mean corpuscular hemoglobin; MCHC: mean corpuscular hemoglobin concentration. Data are expressed as mean ± standard deviation.n = 6 (3/sex); the values in the same line for a given sex followed by different letters in exponent are significantly different (p<0.05) according to the Newman–Keuls multiple comparison test.


### 3.2.4. Urinary Biochemical Parameters

Values of parameters evaluated in the urine of animals are summarized in Table4. In females, there were a decrease in the creatinine level (all doses, p<0.05) and an increase in protein levels (500 and 1000 mg/kg, p<0.001). However, there was no significant difference (p>0.05) for urea between the treated and control females. In males, no significant difference (p>0.05) was observed for creatinine, while urea (p<0.001) and protein (p<0.01) increased at the doses 500 and 1000 mg/kg b.w.Table 4
Effect of oral administration of thePsidium guajava extract on urinary biochemical parameters.
Parameters
Females
Males
Control
250 mg/kg
500 mg/kg
1000 mg/kg
Control
250 mg/kg
500 mg/kg
1000 mg/kg
Creatinine (mg/dL)
53.07 ± 0.832c
42.00 ± 0.400a
38.80 ± 3.555a
48.53 ± 1.007b
64.13 ± 3.449a
63.87 ± 1.890a
63.87 ± 2.663a
58.40 ± 6.053a
Urea (mg/dL)
1921 ± 184.2ab
1618 ± 197.4a
1877 ± 84.59ab
2066 ± 118.4b
1579 ± 105.3a
1482 ± 99.63a
4355 ± 171.1c
2649 ± 30.39b
Proteins (g/dL)
0.2721 ± 0.042a
0.1837 ± 0.020a
0.7211 ± 0.135b
1.020 ± 0.040c
1.136 ± 0.112a
1.252 ± 0.023ab
1.306 ± 0.035b
1.395 ± 0.042b
Data are expressed as mean ± standard deviation.n = 8 (4/sex); the values in the same line for a given sex followed by different letters in exponent are significantly different (p<0.05) according to the Newman–Keuls multiple comparison test.


### 3.2.5. Serum Biochemical Parameters

Investigations made on the animal’s sera made it possible to obtain data in Table5. In females, there was a decrease in HDL cholesterol levels (500 and 1000 mg/kg, p<0.05), triglycerides (p<0.01), total protein (p<0.001), and creatinine (p<0.05) at all doses. AST levels (p<0.01), total cholesterol (p<0.001), LDL cholesterol (p<0.01), and urea (p<0.01) increased for all doses of the extract compared to those in control animals, while ALT levels showed no significant difference. In males, there was no significant difference between triglyceride levels of the test animals and control animals (p>0.05). Total protein levels (500 and 1000 mg/kg, p<0.001), ALT (p<0.01), AST, total cholesterol, and LDL cholesterol (p<0.001) decreased significantly at all doses, while urea (1000 mg/kg, p<0.01), HDL cholesterol (p<0.01), and creatinine (p<0.001) increased for all extract doses.Table 5
Effect of oral administration of thePsidium guajava extract on serum biochemical parameters.
Parameters
Females
Males
Control
250 mg/kg
500 mg/kg
1000 mg/kg
Control
250 mg/kg
500 mg/kg
1000 mg/kg
ALT (U/I)
66.20 ± 1.968a
64.75 ± 2.404a
70.36 ± 5.099a
68.33 ± 1.758a
89.57 ± 2.362c
74.21 ± 3.148b
42.49 ± 5.613a
41.47 ± 3.573a
AST (U/I)
46.27 ± 2.310a
71.88 ± 1.817c
64.02 ± 2.667b
58.46 ± 5.535b
79.44 ± 0.873c
66.06 ± 2.197a
69.84 ± 1.155b
65.85 ± 1.671a
Total cholesterol (mg/dL)
47.14 ± 2.277a
62.99 ± 2.364c
61.27 ± 0.735c
52.70 ± 1.767b
90.69 ± 2.414d
64.79 ± 4.614b
53.19 ± 1.068a
70.51 ± 1.258c
HDL cholesterol (mg/dL)
35.72 ± 0.705b
34.88 ± 5.009b
26.57 ± 1.301a
21.04 ± 3.239a
31.74 ± 2.260a
42.24 ± 4.495b
49.80 ± 4.061c
56.92 ± 1.596d
LDL cholesterol (mg/dL)
22.25 ± 2.873a
38.32 ± 7.160b
44.58 ± 0.7714b
40.99 ± 4.274b
69.70 ± 0.777d
32.84 ± 6.584c
14.16 ± 4.669a
24.42 ± 0.809b
Triglycerides (mg/dL)
54.14 ± 0.885c
51.02 ± 1.113b
49.37 ± 0.692b
46.71 ± 1.113a
53.77 ± 0.692a
51.48 ± 1.262a
53.87 ± 1.301a
54.14 ± 1.113a
Creatinine (mg/dL)
1.036 ± 0.045c
0.571 ± 0.054b
0.631 ± 0.045b
0.488 ± 0.023a
0.4762 ± 0.038a
0.7738 ± 0.045b
0.8929 ± 0.071c
1.048 ± 0.086d
Urea (mg/dL)
69.91 ± 4.243a
82.41 ± 6.263b
90.28 ± 1.389b
84.26 ± 1.604b
70.83 ± 1.389a
74.07 ± 5.782a
78.70 ± 2.122a
89.81 ± 5.613b
Total proteins (g/dL)
4.926 ± 0.071c
4.192 ± 0.112b
4.072 ± 0.124b
3.501 ± 0.023a
3.426 ± 0.141b
3.401 ± 0.117b
3.015 ± 0.069a
2.878 ± 0.118a
ALT: alanine aminotransferase; AST: aspartate aminotransferase. Data are expressed as mean ± standard deviation.n = 8 (4/sex); the values in the same line for a given sex followed by different letters in exponent are significantly different (p<0.05) according to the Newman–Keuls multiple comparison test.


### 3.2.6. Histological Studies

Histopathological examinations were performed on the kidney and liver of animals to verify whether they were damaged by the daily administration of theP. guajava methanolic bark extract; different observations are illustrated. In females (Figure 5), the liver appeared normal at doses 250 and 500 mg/kg b.w., but at the dose 1000 mg/kg, there was inflammation (infiltration of leucocytes). The kidney, on the contrary, showed no alteration although color changes were observed. However, in male rats (Figure 6), the liver as well as the kidney showed no damage at all dose levels tested.Figure 5
Liver (L) and kidney (K) sections of female rats showing effects of oral administration of thePsidium guajava extract over 28 days; L0, K0: control group; L1, K1: 250 mg/kg; L2, K2: 500 mg/kg; L3, K3: 1000 mg/kg. Indicators: A, branch of the hepatic portal vein; B, hepatocytes; c, leukocyte infiltration inflammation; s, sinusoid; v, centrolobular vein; G, glomerulus; EU, urinary space.
(a)
(b)
(c)
(d)
(e)
(f)
(g)
(h)Figure 6
Liver (L) and kidney (K) sections of male rats showing effects of oral administration of thePsidium guajava extract over 28 days; L0, K0: control group; L1, K1: 250 mg/kg; L2, K2: 500 mg/kg; L3, K3: 1000 mg/kg. Indicators: A, branch of the hepatic portal vein; B, hepatocytes; s, sinusoid; v, centrolobular vein; G, glomerulus; EU, urinary space; Ep, hepatic portal space; Cb, bile duct; P, proximal convoluted tubule.
(a)
(b)
(c)
(d)
(e)
(f)
(g)
(h)


## 3.2.1. Weight Growth and Food Intake

Body weight changes in females treated at different doses of the extract during the four weeks of the study presented an almost constant body weight compared to those in the control whose evolution was growing (Figure1). On the contrary, in male rats treated with the P. guajava methanolic bark extract, body weight slightly increased (Figure 2). All female and male animals that received the plant extract had a significantly lower (p<0.05) food intake (Figures 3 and 4).Figure 1
Effect of oral administration of thePsidium guajava extract on body weight changes of female rats during 28 days of the subacute toxicity study.Figure 2
Effect of oral administration of thePsidium guajava extract on body weight changes of male rats during 28 days of the subacute toxicity study.Figure 3
Effect of oral administration of thePsidium guajava extract on food intake of female rats during 28 days of the subacute toxicity study. ∗indicates significant values at p<0.05.Figure 4
Effect of oral administration of thePsidium guajava extract on food intake of male rats during 28 days of the subacute toxicity study. ∗indicates significant values at p<0.05.


## 3.2.2. Relative Weight of Organs

Relative weight values of organs of all animals are presented in Table2. In females, the liver and spleen decreased at all extract doses (p<0.001), while the kidney, heart, and lungs significantly increased (p<0.001) in weight compared to those in control rats. In males, decreased weight of the liver, spleen, and lung (all doses, p<0.001) and heart (500 and 1000 mg/kg, p<0.001) of animals treated with the plant extract has been observed; relative weight of the kidney showed no significant difference from that of the control.Table 2
Effect of oral administration of thePsidium guajava extract on the relative weight of organs and weight gain.
Parameters
Females
Males
Control
250 mg/kg
500 mg/kg
1000 mg/kg
Control
250 mg/kg
500 mg/kg
1000 mg/kg
Liver
3.364 ± 0.077c
2.928 ± 0.026b
2.962 ± 0.037b
2.842 ± 0.001a
3.276 ± 0.133c
2.770 ± 0.049b
2.491 ± 0.002a
2.694 ± 0.057b
Kidney
0.573 ± 0.007a
0.612 ± 0.001b
0.610 ± 0.005b
0.662 ± 0.0005c
0.632 ± 0.015a
0.633 ± 0.002a
0.633 ± 0.001a
0.630 ± 0.007a
Heart
0.301 ± 0.004a
0.362 ± 0.0006c
0.355 ± 0.002c
0.326 ± 0.007b
0.324 ± 0.002c
0.320 ± 0.006c
0.271 ± 0.006a
0.285 ± 0.002b
Lung
0.534 ± 0.002a
0.666 ± 0.005b
0.710 ± 0.003d
0.700 ± 0.005c
0.725 ± 0.010d
0.593 ± 0.022b
0.546 ± 0.003a
0.637 ± 0.0009c
Spleen
0.472 ± 0.006d
0.341 ± 0.007a
0.370 ± 0.017b
0.404 ± 0.005c
0.509 ± 0.038c
0.463 ± 0.006b
0.397 ± 0.005a
0.374 ± 0.001a
Weight gain (g)
65.2
−2.9
1.5
−2.7
60.1
12.5
26.5
19.2
Data are expressed as mean ± standard deviation.n = 8 (4/sex); the values in the same line for a given sex followed by different letters in exponent are significantly different (p<0.05) according to the Newman–Keuls multiple comparison test.


## 3.2.3. Hematological Parameters

Administration of theP. guajava methanolic bark extract to animals increases (p<0.05) the red blood cell number and reduces the amount of platelets in females (1000 mg/kg). In males (1000 mg/kg), it resulted in an increase (p<0.01) in the platelet number. However, the other parameters showed no significant difference (p>0.05) at all tested doses compared to those in control animals. All of these data are presented in Table 3.Table 3
Effect of oral administration of thePsidium guajava extract on hematological parameters.
Parameters
Females
Males
Control
250 mg/kg
500 mg/kg
1000 mg/kg
Control
250 mg/kg
500 mg/kg
1000 mg/kg
WBC (103/μL)
7.05 ± 1.17a
9.16 ± 1.63a
6.36 ± 1.38a
10.05 ± 2.98a
5.30 ± 2.21a
6.40 ± 1.25a
9.46 ± 0.93a
7.95 ± 2.67a
Lymphocytes (%)
41.58 ± 3.03a
34.25 ± 5.85a
36.23 ± 0.97a
36.85 ± 2.52a
34.40 ± 5.65a
37.42 ± 3.27a
36.17 ± 3.87a
38.72 ± 1.53a
Monocytes (%)
7.63 ± 1.92a
7.76 ± 1.65a
7.13 ± 0.30a
11.30 ± 2.43a
8.36 ± 1.57a
6.36 ± 0.76a
8.20 ± 3.47a
9.23 ± 3.75a
Granulocytes (%)
9.20 ± 4.18a
23.73 ± 10.08a
20.40 ± 2.21a
15.00 ± 3.00a
23.10 ± 9.80a
18.80 ± 6.90a
19.47 ± 4.32a
13.33 ± 1.90a
PLT (103/μL)
677.3 ± 30.07b
735.7 ± 16.50b
751.0 ± 9.539b
494.0 ± 76.08a
396.0 ± 10.00a
438.0 ± 47.84a
529.7 ± 30.09a
691.0 ± 108.5b
MPV (fL)
11.83 ± 1.12a
10.87 ± 1.79a
10.93 ± 2.12a
9.46 ± 0.75a
9.83 ± 1.55a
9.13 ± 0.37a
8.80 ± 0.70a
11.00 ± 1.40a
RBC (106/μL)
7.29 ± 0.20a
7.32 ± 0.45a
7.05 ± 0.09a
8.08 ± 0.24b
6.34 ± 1.93a
8.07 ± 0.30a
7.89 ± 0.52a
7.67 ± 0.24a
HGB (g/dL)
16.20 ± 0.10a
16.40 ± 0.90a
15.85 ± 0.05a
16.10 ± 0.40a
16.25 ± 0.05a
17.07 ± 0.61a
17.05 ± 0.15a
17.00 ± 0.20a
HCT (%)
43.50 ± 1.00a
44.45 ± 2.45a
41.50 ± 1.31a
45.20 ± 2.10a
43.25 ± 0.95a
47.03 ± 0.80a
45.53 ± 1.88a
45.07 ± 4.53a
MCV (fL)
62.60 ± 3.98a
57.73 ± 2.80a
58.93 ± 1.06a
55.90 ± 3.89a
58.00 ± 0.50a
58.37 ± 1.41a
60.15 ± 0.55a
58.67 ± 4.10a
MCH (pg)
22.70 ± 0.88a
21.50 ± 0.50a
21.87 ± 0.66a
20.33 ± 1.42a
21.75 ± 0.35a
21.13 ± 1.29a
21.20 ± 0.81a
21.53 ± 0.80a
MCHC (g/dL)
37.20 ± 1.10a
37.40 ± 0.95a
37.37 ± 0.66a
36.43 ± 1.65a
37.55 ± 0.95a
36.27 ± 1.80a
36.63 ± 1.01a
36.90 ± 3.57a
WBC: white blood cells; PLT: platelets; MPV: mean platelet volume; RBC: red blood cells; HGB: hemoglobin; HCT: hematocrit; MCV: mean corpuscular volume; MCH: mean corpuscular hemoglobin; MCHC: mean corpuscular hemoglobin concentration. Data are expressed as mean ± standard deviation.n = 6 (3/sex); the values in the same line for a given sex followed by different letters in exponent are significantly different (p<0.05) according to the Newman–Keuls multiple comparison test.


## 3.2.4. Urinary Biochemical Parameters

Values of parameters evaluated in the urine of animals are summarized in Table4. In females, there were a decrease in the creatinine level (all doses, p<0.05) and an increase in protein levels (500 and 1000 mg/kg, p<0.001). However, there was no significant difference (p>0.05) for urea between the treated and control females. In males, no significant difference (p>0.05) was observed for creatinine, while urea (p<0.001) and protein (p<0.01) increased at the doses 500 and 1000 mg/kg b.w.Table 4
Effect of oral administration of thePsidium guajava extract on urinary biochemical parameters.
Parameters
Females
Males
Control
250 mg/kg
500 mg/kg
1000 mg/kg
Control
250 mg/kg
500 mg/kg
1000 mg/kg
Creatinine (mg/dL)
53.07 ± 0.832c
42.00 ± 0.400a
38.80 ± 3.555a
48.53 ± 1.007b
64.13 ± 3.449a
63.87 ± 1.890a
63.87 ± 2.663a
58.40 ± 6.053a
Urea (mg/dL)
1921 ± 184.2ab
1618 ± 197.4a
1877 ± 84.59ab
2066 ± 118.4b
1579 ± 105.3a
1482 ± 99.63a
4355 ± 171.1c
2649 ± 30.39b
Proteins (g/dL)
0.2721 ± 0.042a
0.1837 ± 0.020a
0.7211 ± 0.135b
1.020 ± 0.040c
1.136 ± 0.112a
1.252 ± 0.023ab
1.306 ± 0.035b
1.395 ± 0.042b
Data are expressed as mean ± standard deviation.n = 8 (4/sex); the values in the same line for a given sex followed by different letters in exponent are significantly different (p<0.05) according to the Newman–Keuls multiple comparison test.


## 3.2.5. Serum Biochemical Parameters

Investigations made on the animal’s sera made it possible to obtain data in Table5. In females, there was a decrease in HDL cholesterol levels (500 and 1000 mg/kg, p<0.05), triglycerides (p<0.01), total protein (p<0.001), and creatinine (p<0.05) at all doses. AST levels (p<0.01), total cholesterol (p<0.001), LDL cholesterol (p<0.01), and urea (p<0.01) increased for all doses of the extract compared to those in control animals, while ALT levels showed no significant difference. In males, there was no significant difference between triglyceride levels of the test animals and control animals (p>0.05). Total protein levels (500 and 1000 mg/kg, p<0.001), ALT (p<0.01), AST, total cholesterol, and LDL cholesterol (p<0.001) decreased significantly at all doses, while urea (1000 mg/kg, p<0.01), HDL cholesterol (p<0.01), and creatinine (p<0.001) increased for all extract doses.Table 5
Effect of oral administration of thePsidium guajava extract on serum biochemical parameters.
Parameters
Females
Males
Control
250 mg/kg
500 mg/kg
1000 mg/kg
Control
250 mg/kg
500 mg/kg
1000 mg/kg
ALT (U/I)
66.20 ± 1.968a
64.75 ± 2.404a
70.36 ± 5.099a
68.33 ± 1.758a
89.57 ± 2.362c
74.21 ± 3.148b
42.49 ± 5.613a
41.47 ± 3.573a
AST (U/I)
46.27 ± 2.310a
71.88 ± 1.817c
64.02 ± 2.667b
58.46 ± 5.535b
79.44 ± 0.873c
66.06 ± 2.197a
69.84 ± 1.155b
65.85 ± 1.671a
Total cholesterol (mg/dL)
47.14 ± 2.277a
62.99 ± 2.364c
61.27 ± 0.735c
52.70 ± 1.767b
90.69 ± 2.414d
64.79 ± 4.614b
53.19 ± 1.068a
70.51 ± 1.258c
HDL cholesterol (mg/dL)
35.72 ± 0.705b
34.88 ± 5.009b
26.57 ± 1.301a
21.04 ± 3.239a
31.74 ± 2.260a
42.24 ± 4.495b
49.80 ± 4.061c
56.92 ± 1.596d
LDL cholesterol (mg/dL)
22.25 ± 2.873a
38.32 ± 7.160b
44.58 ± 0.7714b
40.99 ± 4.274b
69.70 ± 0.777d
32.84 ± 6.584c
14.16 ± 4.669a
24.42 ± 0.809b
Triglycerides (mg/dL)
54.14 ± 0.885c
51.02 ± 1.113b
49.37 ± 0.692b
46.71 ± 1.113a
53.77 ± 0.692a
51.48 ± 1.262a
53.87 ± 1.301a
54.14 ± 1.113a
Creatinine (mg/dL)
1.036 ± 0.045c
0.571 ± 0.054b
0.631 ± 0.045b
0.488 ± 0.023a
0.4762 ± 0.038a
0.7738 ± 0.045b
0.8929 ± 0.071c
1.048 ± 0.086d
Urea (mg/dL)
69.91 ± 4.243a
82.41 ± 6.263b
90.28 ± 1.389b
84.26 ± 1.604b
70.83 ± 1.389a
74.07 ± 5.782a
78.70 ± 2.122a
89.81 ± 5.613b
Total proteins (g/dL)
4.926 ± 0.071c
4.192 ± 0.112b
4.072 ± 0.124b
3.501 ± 0.023a
3.426 ± 0.141b
3.401 ± 0.117b
3.015 ± 0.069a
2.878 ± 0.118a
ALT: alanine aminotransferase; AST: aspartate aminotransferase. Data are expressed as mean ± standard deviation.n = 8 (4/sex); the values in the same line for a given sex followed by different letters in exponent are significantly different (p<0.05) according to the Newman–Keuls multiple comparison test.


## 3.2.6. Histological Studies

Histopathological examinations were performed on the kidney and liver of animals to verify whether they were damaged by the daily administration of theP. guajava methanolic bark extract; different observations are illustrated. In females (Figure 5), the liver appeared normal at doses 250 and 500 mg/kg b.w., but at the dose 1000 mg/kg, there was inflammation (infiltration of leucocytes). The kidney, on the contrary, showed no alteration although color changes were observed. However, in male rats (Figure 6), the liver as well as the kidney showed no damage at all dose levels tested.Figure 5
Liver (L) and kidney (K) sections of female rats showing effects of oral administration of thePsidium guajava extract over 28 days; L0, K0: control group; L1, K1: 250 mg/kg; L2, K2: 500 mg/kg; L3, K3: 1000 mg/kg. Indicators: A, branch of the hepatic portal vein; B, hepatocytes; c, leukocyte infiltration inflammation; s, sinusoid; v, centrolobular vein; G, glomerulus; EU, urinary space.
(a)
(b)
(c)
(d)
(e)
(f)
(g)
(h)Figure 6
Liver (L) and kidney (K) sections of male rats showing effects of oral administration of thePsidium guajava extract over 28 days; L0, K0: control group; L1, K1: 250 mg/kg; L2, K2: 500 mg/kg; L3, K3: 1000 mg/kg. Indicators: A, branch of the hepatic portal vein; B, hepatocytes; s, sinusoid; v, centrolobular vein; G, glomerulus; EU, urinary space; Ep, hepatic portal space; Cb, bile duct; P, proximal convoluted tubule.
(a)
(b)
(c)
(d)
(e)
(f)
(g)
(h)


## 4. Discussion

This study was conducted to prevent human exposure to potential risks associated with the use ofP. guajava. In the acute oral toxicity, no abnormality or mortality was observed in rats at the dose of 5000 mg/kg b.w.; thus, the LD50 of the P. guajava methanolic bark extract is greater than 5000 mg/kg. This extract is therefore relatively nontoxic since substances with LD50 between 2000 mg/kg and 5000 mg/kg orally are considered to have low toxicity, but they, under certain conditions, can be dangerous for vulnerable populations [18]. This study goes in the same direction as that conducted by many researchers such as Sekhar et al. [21] who showed that the P. guajava aqueous bark extract has an LD50 greater than 5000 mg/kg and that animals remain healthy after 10 days of additional observation. Roy et al. [10] showed that the P. guajava leaf extract administered at a dose of 2000 mg/kg has an LD50 greater than this dose. Atik et al. [22] showed that the P. guajava ethanolic fruit extract administered in mice has an LD50 greater than 5000 mg/kg b.w.In order to evaluate the effects that could result from repeated use of this plant extract, its subacute toxicity was carried out. It should be noted that body weight is an important parameter that can control the health status of an animal; weight loss is frequently used as the first indicator of the harmful effects of drugs [22]. A substance is considered toxic if it causes a mass reduction of more than 10%, and this condition may be considered a sign of toxicity even if other changes do not occur [23]. Animals’ weight loss during subacute toxicity can be attributed to antinutritional substances such as tannins and saponins present in this plant. These substances have been reported to cause malabsorption of nutrients in the organism [24]. These antinutrients would be responsible for the low intake of food and thus the reduction in body weight of animals treated at different doses of the extract compared to controls. These results corroborate those of Onyekwe et al. [25] who showed progressive weight loss in animals treated with the ethanolic extract of P. guajava roots for 90 days. However, in this study, the weight loss is less than 2%, showing that this plant was not toxic.The evaluation of hematological parameters provides valuable information on the side effects of foreign substances with respect to the hematopoietic system [26] because this system is one of the most susceptible targets for toxic compounds, especially in the bone marrow where red blood cells are produced [27]. The increased amount of red blood cells in females and platelets in males at 1000 mg/kg b.w. indicates that this plant could possess antianemia property [28] and immunomodulatory effect [29]. Although the specific mechanism through which this extract increases these hematological indices was not ascertained in this study, this effect is assumed to be a direct action of the extract on the hematopoietic system. The nonsignificant difference of the other parameters (hemoglobin, hematocrit, etc.) with respect to the control indicates that the blood elements have not been damaged and that the blood’s ability to transport oxygen to the tissues has been preserved during this study. This extract therefore does not affect the hematopoietic system. Uboh et al. [30] have shown that the aqueous P. guajava leaf extract administered in male and female rats for 30 days has a hematopoietic potential. The increased amount of white blood cells observed in this study at 1000 mg/kg b.w. in females could therefore explain the inflammation of the liver shown by histological analysis.Transaminases are enzymatic biomarkers that can indicate tissue damage caused by chemical compounds before structural damage could be observed by conventional histological techniques [31]. These enzymes are synthesized in the cytoplasm and released into the general circulation when the cells are damaged [32] and at some level can be used to verify the extent of hepatocellular damage [33]. In females, the extract selectively affected transaminases; the AST level increased significantly, while the ALT level of the treated animals was not significantly different from that of controls. Since ALT activity is more sensitive to hepatocyte integrity than that of AST [34], this increase in the serum level of AST may be due to the alteration of another organ. However in males, the decrease in ALT and AST levels suggests that this plant extract acts on the liver. Investigations carried by Uboh et al. [30] showed a decrease in transaminase levels in males treated with the aqueous leaf extract of P. guajava and an increase in the AST level in females; they also reported a nonsignificant effect of the aqueous extract of P. guajava leaves on the liver function enzymes and the morphological architecture of the liver tissues. These results reinforce the hypothesis that the inflammation observed in our study is not related to the increase in the serum level of AST. This decrease in transaminase levels may also reflect the hepatoprotective effect of this plant extract. The work of Roy et al. [10] has also shown the hepatoprotective effect of the aqueous extract of P. guajava leaves, with a more pronounced effect at a dose of 500 mg/kg b.w. in both sexes.The liver and kidney are target organs of toxic chemical compounds because of their essential functions in the processes of detoxification and excretion; these organs are very useful in toxicity studies because of their sensitivity to dangerous compounds [35]. In this work, the indices that assessed renal function were creatinine and urea levels. The kidney regulates the excretion of urea and the reabsorption of electrolytes into the blood; when impaired glomerular function occurs, normally excreted substances such as urea and creatinine accumulate in body fluids [29]. Abnormally high serum levels of creatinine, uric acid, and urea are biomarkers of possible kidney malfunction [36]. In our study, although serum levels of urea and creatinine increased, the urinary levels of these two parameters are normal compared to those in the control. Moreover, the sections of the animal’s kidneys showed no alteration of this organ, indicating that the kidneys play their excretory role well. The increase in the serum urea level following administration of the methanol extract of P. guajava may be due to increased protein catabolism. Hence, the relative weight of the kidney could be explained by the hyperactivity of the kidney to detoxify the body.The decrease in the level of serum proteins observed in the animals treated at doses 500 and 1000 mg/kg b.w. and the increase in the urinary protein level at these same doses suggest that the extract would have interfered with the synthesis balance and degradation of total proteins; such decreases can result in hydration that is detrimental to cellular homeostasis.The increase in total cholesterol and LDL cholesterol and the decrease in HDL cholesterol and triglycerides observed in female rats are an indication of a disturbance of lipid metabolism following the administration of this extract to animals. TheP. guajava methanolic bark extract could therefore have hyperlipidemic property. This disturbance could lead to risks of cardiovascular diseases because LDL is responsible for the development of fatty deposits and atheromatous plaques on the arteries [37].


## 5. Conclusion

This study provides information on the toxicological profile of the methanol extract of thePsidium guajava bark; the results obtained demonstrate that the single dose of 5000 mg/kg b.w. of this plant extract administered orally is not toxic. After long-term treatment (28 days) at high doses (1000 mg/kg b.w.), the toxic effects observed were sex specific and the plant may have some hematological potency and hepatoprotective activity with mild organ toxicity.



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*Source: 8306986-2019-12-11.xml*