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Introduction
Stems, leaves and fruits of several Randia genus species contain compounds such as 1-nitropyrene, linoleic acid, palmitic acid, sterols, β-sitosterol, β-sitosterol, campesterol, oleanolic acid acetate, oleanolic acid-3-α-L-arabinoside, and mesembryanthemoidigenic acid, among others, which have been shown to confer biological, antioxidant and various pharmacological properties (Cano-Campos et al., 2011; Kandimalla et al., 2016; Lapikanon et al., 1983). The Randia monantha Benth fruit, known as a “crucetillo” because of the cross shape in which the thorns are presented, is used as an antivenom beverage against poisonous animals and to which various pharmacological properties are attributed, since it is used as an anticancer, antidiabetic, antidiarrheal, to relieve stomachache and many other diseases (Gallardo-Casas et al., 2012). Currently, the fruit is used in various liquors, predominantly: brandy, ethanol, and Jerez wine. However, it has been reported that the aqueous extract of the pulp and the seed of Randia monantha Benth have a higher concentration of polyphenolic compounds, such as chlorogenic acid, rutin, and 4-coumaric acid, which could contribute to their antioxidant activity and other attributed beneficial properties (Juárez-Trujillo et al., 2018). On the other hand, there are reports that polyphenol compounds have antimicrobial activity, but there is little information in the literature about the inhibitory capability of Randia monantha on pathogenic bacteria. Thus, nowadays people consume it in large quantities and for long periods, considering that the administered dose depends on the bite or sting of the poisonous animal, or the severity of the disease (Mendez and Hernández, 2009). Although in tests conducted with saline artemia there are no toxic effects reported in this species, and as far as we know, there are no studies on its acute toxicity in experimental animals. Therefore, the objective of this study was to evaluate the acute toxicity of the aqueous extract of the Randia monantha fruit in a murine model (male Wistar rats) on behavioral parameters, macroscopic evaluation of liver and spleen, analysis of the pancreatic Langerhans islets, and a histopathological examination of liver, kidney, and pancreas tissue. In addition, we evaluated the effect of the aqueous extract at different concentrations on pathogenic bacteria.
Material and Methods
Obtaining the fruits
To obtain the sample, a batch of 100 fruits (5.73 kg) was selected at random, with a maturity index of 26.60. The fruits were collected at the 2018 January-March period in the town of Actopan, Veracruz, located at the latitude: 19°27´36´´, longitude: 96°49´48´´ west 1060 m.a.s.l. After harvest, fruits were washed and disinfected with 0.01% sodium hypochlorite. Manually obtained pulp (1.04 kg) was lyophilized until it became a powder. The taxonomic identification of the fruit was confirmed by biologist Manuel Carlos Durán Espinosa. A copy of the reference fruit, with Num. of herbarium copy: NJuárezT2 (XAL) was deposited in the Xalapa herbarium of the Institute of Ecology A. C (Juárez-Trujillo et al., 2018).
Preparation of experimental animals
The experimental animals used were 21 male Wistar rats, weighing 300 - 359 g and between 3 to 5 months of age from of Neuroethology laboratory of the Universidad Veracruzana. Six groups of n=3 rats corresponding to the different concentrations (10, 100, 1000, 1600, 2900, and 5000 mg/kg weight body) and a control group (without administration) were randomly formed. The rats were housed in transparent plastic boxes, three rats per box inside the bioterium, with light / dark cycles of 12:12 h at a room temperature of 25 ± 2 ºC, and a humidity between 50 - 60 %. The experimental animals carried a standard diet with water and pelletized food ad libitum. They were allowed seven days for acclimatization before the commencement of the research. In this study, all animals were handled in accordance with the guidelines for the Care and Use of Laboratory Animals of Universidad Veracruzana based in the National Institutes of Health.
Acute toxicity study
The dried pulp was mixed at different concentrations with a solution of tween 20 (5%), propylene glycol (5%), and purified water. Doses were prepared according to guide 423, the Organization of Economic Co-operation and Development (OECD, 2001). The groups were administered with extract by intragastric cannulation in a single dose of 5000, 2900, 1600, 1000, 100, and 10 mg/kg body weight for each of the treatment groups, respectively, while the control group received only tween 20, propylene glycol and water used as a vehicle. LD50 was estimated according to the guideline 423 from Organization of Economic Co-operation and Development (OECD, 2001).
The animals were monitored during the first 24 hours after cannulation with special emphasis on the first 30 minutes, and with 15-minute videos taken at 2, 4, 6, 8, 12, and 24 hours in search of apparent modification of toxicological behavior (Cavanaugh, 2003). The symptoms analyzed were mucus, ataxia, salivation, diarrhea, seizures, respiratory disorders, hyperactivity, and paralysis (Pérez, 2017). The weight of the rats was recorded daily for 15 days. On day 15, all rats used in the experimental procedures were sacrificed with an overdose of sodium pentobarbital (60 mg/kg).
A blood sample was obtained by cardiac puncture and collected in plastic tubes containing EDTA. Erythrocyte count, hemoglobin, hematocrit, mean corpuscular volume, mean corpuscular hemoglobin, mean corpuscular hemoglobin concentration, platelet count, and leukocyte count were evaluated using a veterinary hematology analyzer (Abaxis Vatscan HM2). All rats were sacrificed on day 15 and subsequently perfused and dissected; the extraction and weighing of organs, liver, kidneys, pancreas, and spleen were performed.
Histopathology study
Selected organs, including pancreas, liver, kidney, and spleen of the treated and control animal groups, were fixed in 10 % buffered formalin in labeled bottles and subsequently processed for histological examination. The tissue was processed using an automated tissue processor (Leica 820, Germany) and embedded in blocks of paraffin. Stained tissue sections were examined under a light microscope (Leica DM 750, Germany). Number and area of Langerhans islets in the pancreas were determined in at least five islets per section at a 40x magnification. Full images of the selected Langerhans islets were obtained by photomicrographs with Adobe® Photoshop CS6 software, and their analysis was performed using the ImageJ Java® software in which the area, thickness, density, and volume data of the selected islets were obtained according to the methodology reported by Cavanaugh (2003). The numerical concentration of the cells in the islet was determined by manual counting by differential hematoxylin and eosin phloxine (H&E) staining (Coggehall, 1992; Mohammadi and Naik, 2012).
Antimicrobial activity of the aqueous extract of pulp of Randia monantha fruit
The Randia monantha pulp extract antimicrobial activity was evaluated using the broth dilution method. Active cultures of Listeria monocytogenes (ATCC 19115), Staphylococcus aureus (ATCC 25923), Escherichia coli (ATCC 43895), and Salmonella typhimurium (ATCC 14028) were prepared according to Balouri et al. (2016). For the above, dilutions were prepared at 0.5, 1.0, 1.5 and 2.0 mg/mL in a liquid growth medium, distributed in tubes containing a volume of 2 mL. Subsequently, each tube was inoculated with a microbial inoculum prepared in the same medium, which was previously standardized to the Mcfarland scale of 0.5. Each tube was then shaken and incubated for 24 hours at 37 °C. After 24 h, the absorbance λ= 600 nm was read.
Statistical analysis
Analysis of variance was carried out to evaluate the results found in the different treatments with pulp extracts, and subsequently, the significant differences were assessed using the Fisher test with a level of significance p ≤ 0.05, using Minitab program version 17, States United.
Results
Body and organs weight gain
The results of the acute toxicity study indicated that the aqueous extract of the R. monantha pulp administered orally to rats at doses between 10-5000 mg/kg body weight in acute toxicity testing methodology did not cause death or significant toxicity in test animals, indicating that the LD50 is greater than the doses administered. Table 1 shows that the groups corresponding to an administration of 2900 and 5000 mg/kg body weight presented a slight increase in weight that ranged from 0.25 to 7.94%. However, in relation to the weight of the organs, it was observed that the pancreas, liver, kidneys, and spleen did not show significant differences (p > 0.05) compared to the respective organs of the control treatment.
Table 1 Increase in total body weight and in weight of the important vital organs of each
treatment group.
Tabla 1. Incremento del peso corporal total y aumento
del peso de los órganos vitales importantes de cada grupo de
tratamiento.
| Doses (mg/kg Body weight) | Increased Total Body Weight (%) | Pancreas | Liver | Kidney | Spleen |
| 0 (Control) | 0.05 ± 0.02ª | 2.99 ± 0.85ª | 11.01 ± 0.73ª | 3.34 ± 0.41ª | 1.40 ± 0.19ª |
| 10 | 0.25 ± 0.19ª | 2.74 ± 0.68ª | 10.97 ± 1.33ª | 2.63 ± 0.27ª | 1.13 ± 0.24ª |
| 100 | 1.84 ± 0.86b | 3.34 ± 0.69ª | 10.15 ± 1.76ª | 3.12 ± 0.71ª | 1.31 ± 0.23ª |
| 1000 | 5.73 ± 0.70c | 3.04 ± 0.18ª | 9.97 ± 1.52ª | 2.91 ± 0.83ª | 1.57 ± 0.53ª |
| 1600 | 4.64 ± 0.68c,d | 3.27 ± 1.01ª | 10.69 ± 1.60ª | 2.89 ± 0.24ª | 1.30 ± 0.09ª |
| 2900 | 5.39 ± 0.64d | 3.80 ± 0.69ª | 12.45 ± 2.35ª | 3.04 ± 0.63ª | 1.25 ± 0.18ª |
| 5000 | 7.94 ± 0.87e | 3.74 ± 0.23ª | 11.79 ± 2.34ª | 2.87 ± 0.30ª | 1.56 ± 0.12ª |
Mean ± SD, n = 3. Different letters in the same column indicate significant differences (p < 0.05).
Behavior observations
Consistent with these results, the different treatment groups showed no significant difference in the evaluated clinical signs (mucus, ataxia, salivation, diarrhea, convulsions, hyperactivity, paralysis, and changes in breathing), compared to the control treatment.
Macroscopic evaluation of organs
External macroscopic analysis of liver, spleen, and pancreas did not reveal any alteration nor signs of toxicity, without visual differences with the control treatment, indicating the absence of toxicity. However, the kidneys of the group corresponding to an administration of 5000 mg/kg body weight showed yellow spots. The kidney histopathology examination revealed that the yellow areas presented a minor or no blood supply, with a notable decrease in erythrocytes and a decrease in blood flow than the kidney cuts of the control treatment (Figure 1).
Figure 1 Kidney histological examination. A) Control treatment and B) Treatment with a dose of
5000 mg/kg of weight. The arrows point out the blood supply.
Figura 1. Examen histologico del rinon de A)
Tratamiento de control y B) Tratamiento con dosis de 5000 mg/kg de
peso. Las flechas senalan el aporte de sangre.
Hematology parameters analysis
The hematology parameters are summarized in Table 2. The hematological analysis of leucocytes, lymphocytes, monocytes, granulocytes, red blood cell, mean cell volume, hemoglobin, hematocrit, mean cell volume, mean corpuscular hemoglobin, erythrosary distribution index, platelets, mean platelet volume, and platelet distribution revealed no significant differences (p > 0.05) in relation to the control treatment and the days analyzed, the values were within the range considered normal (Tasić et al., 2021; Nagamma et al., 2019; Dantas et al., 2006). The samples did not show significant differences (p > 0.05) in the triglycerides and cholesterol concentration with the control group, which should be considered in treating dyslipidemias. Treatments with a dose between 1600 and 5000 mg/kg of body weight exhibited at time zero (first day of administration) had an increase in glucose concentration (116-131.69 mg/dL) compared to the control group (62.79-77.63 mg/dL). However, these values are in the range of values considered normal for laboratory animals (Lapchik et al., 2017).
Table 2 Hematological examination of treatments with different dosis (0-5000 mg/kg) of the
standardized aqueous extract of Randia monantha
pulp at 1, 7 and 15 days of administration.
Tabla 2. Examen hematológico de los tratamientos con
diferentes dosis (0-5000 mg/kg) del extracto acuoso estandarizado de
pulpa de Randia monantha a los 1, 7 y 15 días de
administración.
| Day 1 | Dose (mg/kg weight) | ||||||
| Parameters | Control | 10 | 100 | 1000 | 1600 | 2900 | 5000 |
| Leukocytes (109/L) | 12.97±1.5 | 10.21±2.51 | 16.41±8.15 | 10.24±2.57 | 12.23±3.91 | 14.03±4.18 | 7.77±0.49* |
| Lymphocytes (109/L) | 8.80±0.72 | 7.88±1.43 | 9.62±1.29 | 7.82±2.48 | 8.34±4.15 | 6.42±1.59 | 5.59±1.26* |
| Monocytes (109/L) | 0.53±0.34 | 0.36±0.02 | 0.75±0.08 | 0.56±0.01 | 0.18±0.01 | 0.29±0.10 | 0.27±0.23 |
| Lymphocytes (%) | 68.00±2.40 | 76.16±3.26 | 64.96±19.11 | 75.33±7.03 | 64.93±15.34 | 49.70±26.3 | 71.56±11.42 |
| Monocytes (%) | 4.00±2.26 | 2.80±0.91 | 3.46±0.45 | 0.60±0.00 | 0.63±0.05 | 2.10±0.14 | 2.73±0.10 |
| Granulocytes (%) | 28.00±0.14 | 21.03±4.38 | 31.56±5.80 | 24.10±7.00 | 34.43±5.29 | 48.20±26.16 | 25.73±9.57 |
| Red blood rell (1012/L) | 9.07±0.35 | 9.00±0.27 | 8.35±1.69 | 9.04±0.31 | 8.71±0.12 | 9.86±0.01 | 9.42±0.83 |
| Hemoglobin (g/dL) | 17.50±0.00 | 17.40±0.30 | 16.10±2.87 | 17.36±0.20 | 16.76±0.81 | 19.30±0.56 | 18.43±0.55 |
| Hematocrit (%) | 52.69±1.25 | 53.54±1.66 | 50.75±9.51 | 55.27±1.95 | 51.19±3.89 | 55.66±1.37 | 51.84±3.83 |
| Mean cell volumen (fl) | 58.50±3.53 | 59.33±0.57 | 60.66±1.52 | 61.33±0.57 | 58.66±3.78 | 56.50±0.70 | 55.00±2.64 |
| Mean corcuspular hemoglobin (g/dL) | 33.15±0.77 | 32.56±0.49 | 31.80±0.36 | 31.43±0.87 | 32.83±0.92 | 34.60±0.14 | 35.66±1.76 |
| Erythrosary distribution index (%) | 17.75±0.49 | 16.70±0.20 | 18.10±1.31 | 16.50±0.40 | 18.60±0.95 | 16.45±0.35 | 16.60±0.96 |
| Platelets (109/L) | 620.50±47.37 | 587.00±60.62 | 504.66±31.07 | 754.33±127.44 | 814.33±88.66 | 493.00±15.36 | 20.66 ± 2.03* |
| Mean platelet volumen (fl) | 6.65±0.35 | 6.56±0.11 | 6.66±0.15 | 6.63±0.11 | 6.76±0.41 | 6.90±0.42 | 6.63±0.37 |
| Triglycerides (mg/dL) | 100.19±27.83 | 95.80±31.88 | 95.80±14.71 | 62.67±5.53 | 104.99±18.60 | 143.11±45.62 | 73.83±27.29 |
| Glucose (mg/dL) | 52.79±12.29 | 57.89±16.12 | 54.52±15.44 | 74.47±7.89 | 104.23±36.60* | 110.40±23.70* | 124.08±23.83* |
| Cholesterol (mg/dL) | 80.19±5.80 | 76.90±6.98 | 91.40±10.44 | 75.36±9.05 | 89.66±12.83 | 76.08±9.83 | 86.95±21.16 |
| Day 7 | |||||||
| Leocucytes (109/L) | 15.57±4.62 | 10.39±2.26 | 16.41±8.15 | 10.24±2.57 | 12.23±3.92 | 10.35±1.01 | 14.38±7.82 |
| Lymphocytes (109/L) | 9.20±0.86 | 7.88±1.43 | 9.62±1.29 | 7.81±2.49 | 8.34±4.15 | 7.78±0.32 | 5.47±1.18* |
| Monocytes (109/L) | 0.49±0.25 | 0.26±0.02 | 0.75±0.09 | 0.16±0.01 | 0.18±0.01 | 0.13±0.02 | 0.48±0.05 |
| Lymphocytes (%) | 61.40±11.55 | 76.16±3.26 | 64.96±19.11 | 75.33±7.03 | 64.93±15.34 | 75.75±10.53 | 247.63±73.57 |
| Monocytes (%) | 3.30±0.20 | 2.80±0.91 | 3.46±0.45 | 0.60±0.00 | 0.63±0.05 | 1.30±0.98 | 2.96±0.59 |
| Granulocytes (%) | 35.26±12.58 | 21.03±4.38 | 31.56±15.80 | 24.10±7.00 | 34.43±15.29 | 23.00±9.45 | 49.43±6.27 |
| Red blood rell (1012/L) | 8.80±0.52 | 9.00±0.27 | 8.35±1.69 | 9.04±0.31 | 8.71±0.12 | 9.62±0.35 | 9.74±0.48 |
| Hemoglobin (g/dL) | 17.00±0.86 | 17.40±0.30 | 16.10±2.87 | 17.36±0.20 | 16.76±0.81 | 18.75±1.34 | 18.33±0.73 |
| Hematocrit (%) | 50.01±4.73 | 53.54±1.66 | 50.75±9.51 | 55.27±1.95 | 51.19±3.89 | 53.46±4.47 | 51.99±3.20 |
| Mean cell volumen (fl) | 57.00±3.60 | 59.33±0.57 | 60.66±1.52 | 61.33±0.57 | 58.66±3.78 | 55.50±2.12 | 53.66±2.08 |
| Mean corcuspular hemoglobin (g/dL) | 34.03±1.62 | 32.56±0.49 | 31.8±0.36 | 31.43±0.87 | 32.83±0.92 | 35.1±0.56 | 35.23±0.80 |
| Platelets (109/L) | 816.00±34.02 | 587.00±60.62 | 504.66±31.80 | 754.33±124.44 | 814.33±88.66 | 896.50±255.36 | 304.66±67.68* |
| Platelet (%) | 0.53±0.02 | 0.38±0.04 | 0.33±0.02 | 0.50±0.09 | 0.55±0.09 | 0.62±0.14 | 0.14±0.02* |
| Platelet distribution width (%) | 31.03±0.28 | 32.16±0.45 | 31.66±0.95 | 31.53±0.57 | 31.70±0.86 | 32.6±0.56 | 33.83±3.35 |
| Triglycerides (mg/dL) | 90.61±24.95 | 113.77±21.85 | 135.32±4.79 | 113.97±41.00 | 103.11±7.85 | 85.62±14.39 | 73.05±7.28 |
| Glucose (mg/dL) | 59.00±19.75 | 82.14±10.44 | 60.51±16.15 | 65.96±15.15 | 67.63±14.05 | 128.36±10.63* | 116.00±20.22* |
| Cholesterol (mg/dL) | 47.09±11.17 | 52.90±11.11 | 50.20±4.66 | 50.87±9.00 | 48.85±2.44 | 44.93±1.71 | 39.67±7.49 |
| Day 15 | |||||||
| Leocucytes (109/L) | 15.30±2.95 | 10.05±1.79 | 12.41±0.60 | 11.11±1.18 | 12.73±5.12 | 10.14±0.71 | 8.01±3.29* |
| Lymphocytes (109/L) | 9.89±1.28 | 7.01±0.19 | 9.08±0.50 | 8.25±1.69 | 7.78±3.57 | 8.53±0.73 | 5.58±2.21 |
| Monocytes (109/L) | 0.48±0.02 | 0.19±0.03 | 0.25±0.01 | 0.45±0.06 | 0.44±0.01 | 0.05±0.00 | 0.22±0.02 |
| Lymphocytes (%) | 65.86±11.61 | 70.90±10.12 | 73.40±7.29 | 73.76±8.21 | 60.56±11.21 | 84.10±1.27 | 70.03±7.03 |
| Monocytes (%) | 3.06±0.92 | 1.66±0.84 | 1.96±0.36 | 4.26±0.80 | 4.36±6.43 | 0.60±0.00 | 4.56±0.78 |
| Granulocytes (%) | 30.40±10.98 | 27.43±8.30 | 24.63±4.93 | 21.96±4.50 | 35.06±5.65 | 15.40±127 | 25.46±8.98 |
| Red blood rell (1012/L) | 8.80±0.33 | 9.91 0.13 | 10.10±0.74 | 8.94±0.41 | 8.86±0.09 | 8.98±0.55 | 9.00±0.49 |
| Hemoglobin (g/dL) | 16.06±0.66 | 19.16±0.36 | 19.26±0.96 | 18.53 0.87 | 17.50±1.01 | 17.40±0.56 | 17.36±0.66 |
| Hematocrit (%) | 49.81±2.52 | 59.36±0.61 | 59.99±4.18 | 56.42±2.57 | 53.74±2.54 | 49.19±1.56 | 49.03±2.56 |
| Mean cell volumen (fl) | 56.66±2.08 | 60.00±0.00 | 59.33±1.52 | 63.00±0.00 | 60.66±3.05 | 55.00±1.41 | 54.33±2.30 |
| Mean corcuspular hemoglobin (g/dL) | 32.26±1.92 | 19.33±0.25 | 19.10±0.45 | 32.80±0.69 | 32.50±1.44 | 35.45±0.07 | 35.36±0.90 |
| Platelets (109/L) | 451.33±46.98 | 586.66 101.07 | 552.66±55.62 | 625.33 87.84 | 566.00±40.18 | 951.00±178.00 | 504.33±83.31 |
| Platelet (%) | 0.29±0.02 | 0.38±0.05 | 0.37±0.03 | 0.43 0.06 | 0.39±0.08 | 0.65±0.11 | 0.34±0.06 |
| Platelet distribution width (%) | 33.66±4.43 | 32.13±0.90 | 32.46±0.23 | 32.00±0.23 | 32.43±0.87 | 32.40±0.28 | 32.16±0.45 |
| Triglycerides (mg/dL) | 89.02±32.93 | 113.83±41.49 | 121.33±21.28 | 113.25±40.87 | 105.74±34.24 | 121.49±21.17 | 89.34±49.82 |
| Glucose (mg/dL) | 67.63±29.75 | 59.88±19.67 | 66.14±16.32 | 66.14±16.32 | 80.94±2.11 | 128.94±17.74* | 131.69±20.20* |
| Cholesterol (mg/dL) | 44.49±6.06 | 60.69±7.22 | 48.74±11.06 | 61.32±11.96 | 53.14±6.54 | 42.21±3.66 | 39.62±2.35 |
Values are expressed as mean ± SD, n=3.
*Significant differences with the control treatment, p < 0.05.
Histopathology examination of pancreatic cells
The size, area, diameter, and volume of the Langerhans islet of the different treatments did not show significant differences (p > 0.05) (Table 3), which is important since its parameters directly influence the maintenance of glucose in the blood and their alteration is related to diabetes (Jo et al., 2007). However, a significantly lower concentration of langerhans islets (643 cell/mm2) was observed in the 5000 mg/kg body weight treatment with compared to the other treatments. The groups that were administered 10, 100, 1000, 1600 and 2900 mg/kg body weight of extract exhibited a concentration of cells in the Langerhans islets that varied from 2008 to 3146 cells/mm2, without significant differences (p > 0.05) with the control treatment (3100 cells/mm2). In contrast, the treatment of 5000 (643 cells / mm2) mg/kg body weight, showed a significant decrease in the number of cells in relation to the control treatment. Consistent with these results, the pancreas cells of the group administered with 5000 mg/kg body weight of extract showed a lower number of cells (alpha, beta, and delta) in the Langerhans islets compared to the control group (Figure 2).
Table 3 Cell concentration and morphological parameters of the Langerhans islets of each of
the treatments.
Tabla 3. Concentración celular y parámetros
morfológicos de los islotes de Langerhans de cada uno de los
tratamientos.
| Groups | Cell concentration / mm2 | Central Islet perimeter (µm) | Central Islet area (µm٢) | Islet diameter (µm) | Cell number |
| (mg/body weight) | |||||
| 0 (Control) | 3146 ± 724b | 2436 ± 552a | 255284 ± 49380ab | 183.3 ± 50.35a | 780 ± 81b,c |
| 10 | 2984 ± 296b | 2225 ± 691a | 274342 ± 11183ab | 233.3 ± 86.24a | 969 ± 57b,c |
| 100 | 2585 ± 322b | 2412 ± 277a | 348403 ± 71744b | 243.3 ± 28.91a | 885 ± 93b,c |
| 1000 | 2622 ± 1000b | 2279 ± 696a | 237570 ± 67146ab | 196.6 ± 5.77a | 621 ± 30b |
| 1600 | 3311 ± 733b | 2013 ± 156a | 230044 ± 23702ab | 210.0 ± 26.53a | 731 ± 88b |
| 2900 | 2008 ± 510b | 1748 ± 228a | 178307 ± 52620a | 186.7 ± 49.37a | 375 ± 158ab |
| 5000 | 643 ± 412a | 2054 ± 489a | 283802 ± 10467ab | 206.7 ± 20.81a | 174 ± 109a |
Mean ± SD, n = 3. Different letters in the same column indicate significant differences (p <0.05).
Figure 2 Pancreas histological examination. A) Control treatment and B) Treatment with a dose
of 5000 mg/kg of weight. The arrows point out the Langerhans beta cells.
Figura 2. Examen histologico del pancreas. A)
Tratamiento de control y B) Tratamiento con dosis de 5000 mg/kg de
peso. Las flechas senalan las celulas beta de Langerhans.
Antimicrobial properties of aqueous extract
The antimicrobial effects of the Randia monantha fruit aqueous extract against Gram (-) and Gram (+) bacteria are shown in Table 4. It can be seen that the aqueous extracts did not present dose-dependent antimicrobial activity in the test concentration range (0.5-2.0 mg/mL) against Gram (+) bacteria, nor Gram (-) test bacteria. These results were similar to those reported for the methanol and ethanol extract of Randia aculeata against Mycobacterium bovis (García-Cruz, 2018), but different from those reported for the crude extract of Randia dumetorum, which exhibited significant antimicrobial activity (Kumar et al., 2006).
Table 4 Antibacterial activity of aqueous extract of Randia monantha fruit.
Tabla 4. Actividad antibacteriana del
extracto acuoso de fruto de Randia
monantha.
| Pathogenic bacteria | Control | Dose (mg/mL) | |||
| 0.5 | 1 | 1.5 | 2 | ||
| Listeria monocytogenes | 0.18 ± 0.01a | 0.28 ± 0.04b | 0.32 ± 0.05a,b | 0.42 ± 0.09b,c | 0.53 ± 0.03c |
| Salmonella typhimurium | 0.59 ± 0.02d | 0.36 ± 0.01ª | 0.41 ± 0.01b | 0.44 ± 0.03bc | 0.47 ± 0.01cd |
| Staphylococcus aureus | 0.33 ± 0.05b | 0.24 ± 0.05a | 0.23 ± 0.03a | 0.37 ± 0.05b | 0.41 ± 0.03b |
| Escherichia coli | 0.21 ± 0.04a | 0.30 ± 0.04b | 0.31 ± 0.05b | 0.39 ± 0.03 b,c | 0.46 ± 0.03c |
Mean ± SD, n = 3. Different letters in the same column indicate significant differences (p <0.05).
Discussion
In accordance with the OECD criteria under the regulation established by the Globally Harmonized System (GHS) and classification labeling for chemical substances and mixtures, substances with LD50 > 5000 mg/kg body weight are category 5 or classified as unclassified (OECD, 2001), suggesting that the concentrations tested in this study do not represent a toxic risk. Similarly, according to Hogde and Sterner (2005), scale compounds with LD50 between 5000-15000 mg/kg body weight are considered as practically non-toxic substances. The test animals showed weight gain directly proportional to the extract concentration administered, which could be due to the high concentration of sugars present in the pulp of Randia monatha Benth (Juarez-Trujillo et al., 2018). This, since the increase in weight regularly relates to alterations in the macronutrient metabolism such as carbohydrates, proteins or fat, and even to hydromineral, or intracellular and extracellular deregulation and these nutrients play an appropriate role in physiological functions (Ezeonwumelu et al., 2011). In turn, it has been reported that changes in the weight of the organs are an important and sensitive parameter for the detection of harmful toxicological effects that are often associated with the administration of a chemical substance toxic to the organism (Sellers et al., 2007). Despite the increase in body weight, the weight of pancreas, liver, kidneys, and spleen did not show a significant increase with respect to the weight of these organs in the control group. This corroborates that the concentrations tested in this study were not toxic to the organism, since an increase in the weight of the organs suggests the appearance of hypertrophy while the decrease suggests atrophy or necrosis in the target organ (Teo et al., 2002).
Hematological parameters are perceptual markers of physiological changes in the health and toxicological status of test animals. The hematological profile suggested no changes in the count of red blood cells, leukocytes, and hemoglobin after the 0, 7, and 15 days of testing compared to the control treatment, suggesting that the aqueous extract has no influence on the hematopoiesis pathway. These results also demonstrated no significant changes in the lipid profile values in the range of tested concentrations, suggesting that this extract does not influence the lipid profile. In turn, the blood test revealed a significant increase in glucose concentration, which is possible since the Randia monantha pulp is rich in reducing sugars and total carbohydrates, which explain the increase in blood glucose and changes in the coloration of Langerhans cells. However, it has been reported that the soluble melanins and antioxidants present in Randia echinocarpa are a potential resource for type II diabetes treatment (Cuevas-Juarez et al., 2014) and that the aqueous extract of Randia nicotica had satisfactory efficacy in glycemic control in diabetic rats (Alamin et al., 2015). It is possible that these differences between the biochemical parameters regarding the antidiabetic effect reported in other species are due to the complexity of the phytochemical compounds present in the aqueous extract.
In general, the human body has several essential organs that are vital and with specific functions, such as liver, pancreas, and kidney, each of them has different functions, either in the metabolism of intake, in the regulation of certain hormones and/or in the excretion of waste products (Umale et al., 2013). Therefore, to know the toxicity of a substance it is essential to know the state of these vital organs. In the histopathological study, we observed that the pancreas and liver in all treated groups showed no changes at the macroscopic and cellular level in comparison to the control. However, the macroscopic observation of kidneys from the treatment administered with high doses revealed yellow spots, and histopathology analysis exhibited a noticeable lesser blood supply in the area corresponding to the yellow spots. There are previous reports indicating that the administration of certain extracts can cause hyperemia, inflammation, and kidney damage since this is an organ responsible for disposing of chemical compounds and is more sensitive to damage by xenobiotic compounds (Saleen et al., 2017; Meilian et al., 2019).
On other hand, oxidative stress, inflammation, and apoptosis have been reported to be responsible for the degeneration of pancreatic islets, which could negatively affect circulating insulin level and result in a persistent hyperglycemia (Nna et al., 2018). All this is a critical parameter to consider in the use of Randia monantha Benth extract, since beta cells are responsible for insulin secretion (Levetan, 2010). Thus, our results suggest that at these concentrations, the oral intake of this extract could have a negative health effect, which is a critical factor in the intake of this fruit, considering that people consume it in excess among other things as a remedy for diabetes.
In turn, the aqueous extract did not have an antimicrobial effect against the test pathogenic bacteria, possibly due to the high content of sugars present in the sample, which are a good source of carbon for the bacteria. However, it is necessary to consider other factors such as the concentration of the extract and the incubation time that affect the bacterial effect of the extracts (Othmen et al., 2020).
Conclusions
According to the hematological parameters obtained in this study, the administration of the R. monantha extract in the range of 10-5000 mg/kg body weight concentrations, does not present acute toxicity considering that there was no death in the different experimental groups, with LD50 greater than the administered dosis, corresponding to class 5 according to the OECD without toxicity. In general, the experimental organisms did not present significant differences in the hematological analyzes with most of the treatments, and all were within the range considered normal. This was consistent with the microscopic analysis in which doses greater than 2900 mg/kg body weight, caused damage to the pancreas, presenting a decrease mainly in beta cells in the Langerhans islets, in addition to generating alteration in blood supply to the kidneys. According to the LD50 and the absence of mortality, the aqueous extract administered orally may indicate that it can be used with some safety degree for diabetes treatment. The microbiological analysis revealed that the extract does not have antimicrobial activity against some pathogenic bacteria and, on the contrary, favors their growth. Therefore, further studies are needed to establish a safe protocol for human clinical trials.
