**TOCOSH FLOUR (***Solanum tuberosum* **L.): A Toxicological Assessment of Traditional Peruvian Fermented Potatoes**

**Jonas Roberto Velasco-Chong <sup>1</sup> , Oscar Herrera-Calderón 1,\* , Juan Pedro Rojas-Armas <sup>2</sup> , Renán Dilton Hañari-Quispe <sup>3</sup> , Linder Figueroa-Salvador <sup>4</sup> , Gilmar Peña-Rojas <sup>5</sup> , Vidalina Andía-Ayme <sup>6</sup> , Ricardo Ángel Yuli-Posadas <sup>7</sup> , Andres F. Yepes-Perez <sup>8</sup> and Cristian Aguilar <sup>9</sup>**


Received: 20 April 2020; Accepted: 29 May 2020; Published: 2 June 2020

**Abstract:** Potato tocosh is a naturally processed potato for nutritional and curative purposes from traditional Peruvian medicine. The aim of this study was to investigate the acute and sub-acute toxicity of tocosh flour (TF). For sub-acute toxicity, TF was administered orally to rats daily once a day for 28 days at doses of 1000 mg/kg body weight (BW). Animals were observed for general behaviors, mortality, body weight variations, and histological analysis. At the end of treatment, relative organ weights, histopathology, hematological and biochemical parameters were analyzed. For acute toxicity, TF was administered orally to mice at doses of 2000 and 5000 mg/kg BW at a single dose in both sexes. Body weight, mortality, and clinical signs were observed for 14 days after treatment. The results of acute toxicity showed that the median lethal dose (LD50) value of TF is higher than 2000 g/kg BW but less than 5000 mg/Kg BW in mice. Death and toxicological symptoms were not found during the treatment. For sub-acute toxicity, we found that no-observed-adverse-effect levels (NOAEL) of TF in rats up to 1000 g/kg BW. There were statistically significant differences in body weight, and relative organ weight in the stomach and brain. No differences in hematological and biochemical parameters were observed when compared with the control group. For sub-acute toxicity, histopathological studies revealed minor abnormalities in liver and kidney tissues at doses of 5000 mg/Kg. Based on these results, TF is a traditional Peruvian medicine with high safety at up to 1000 mg/kg BW for 28 days in rats.

**Keywords:** *Solanum tuberosum* L.; oral toxicity; tocosh; fermented foods; traditional medicine

#### **1. Introduction**

*Solanum tuberosum* L. (Family: Solanaceae) is one of the most important Andean crops, cultivated along the Andean mountain range of South America and spread to other regions worldwide [1]. Over time, Andean farmers have developed frost and drought-resistant crops, which can be planted at heights greater than 3800 m above sea level (m.a.s.l.). In Peru, there are around 3800 varieties of potato and it is one of the main contributors to the world. The potato was domesticated just under 10,000 years ago; staple food crops of the ancient Peruvians not only used fresh potato but also consumed the product in the fermentation state, named tocosh [2].

Tocosh is a naturally processed potato for curative and nutritional purposes, which consists of leaving the potato in pools protected with straw or mesh near a stream for an average of six months, then it is extracted for consumption [3]. At the end of the process, the potato is reduced in size, except for its peel and it gets a very peculiar unpleasant smell. Since Inca and Pre-Incas times, the inhabitants of Ancash, Huánuco, and Junín regions have used tocosh as a medicine, in the form of flour or in its natural state to prepare *mazamorra* (Api in the Quechua language), is the best-known form of consumption (see Figure 1: Elaboration of tocosh). Tocosh flour (TF) is attributed to some beneficial properties such as gastritis, ulcers, gastro-esophagi reflux, and gastric cancer. People consume it by dissolving one teaspoonful per 100 mL in water before food as an alternative treatment. Although tocosh flour consumption is invariable, the normal dose known in traditional medicine is between 500 and 1000 mg/Kg daily (this information was taken according to an interview at the place where potato tocosh was collected).

**Figure 1.** Extraction processing of "potato tocosh" in Yaca district, Huanuco region, Peru.

Potato flour (PF) is characterized by its unpleasant smell, which is the first thing to be perceived, a peculiarity that does not limit its consumption or commercialization, affirming by empirical knowledge that it contains natural penicillin [4] and that among its innumerable benefits it is able to protect the gastric mucosa from damage or inflammation, according to popular customs, this product is used in postpartum, colds, pneumonia, in wound healing, as an antibacterial, healing of hemorrhoids and gastric ulcers, to avoid gastrointestinal infections and mountain sickness [5,6].

′ ″ ′ ″ The potato species (*Solanum tuberosum* L.) are specific products consumed massively, these species present steroidal alkaloids, and when it is not well stored, can cause symptoms of poisoning such as respiratory distress, nausea, vomiting, and diarrhea related to acetylcholinesterase inhibition [7,8]. The primary steroidal glycoalkaloids in potato tubers are R-solanine and R-chaconine, being

−

glycosylated forms of the steroidal alkaloid solanidine, these often improve the flavor of the potato [9]. The concentration of steroidal glycoalkaloids increases in response to several factors, such as injury, fungal attack, poor growing conditions, weather, and unsuitable storage conditions [10]. Nowadays, tocosh flour and derivates are sold as natural products in Peruvian markets but toxicity studies have not been reported to assess its consumption over a long period of time, which could induce any organ damage or death when there is no available data about the correct doses of administration. The main objective of this research was to evaluate the toxicological effect of tocosh flour following the guidelines for subacute and acute oral toxicity in rodents.

#### **2. Material and Methods**

#### *2.1. Collection of Plant Material*

Tocosh was collected in December 2019 from the Amarilis district, crossing the border of Yaca and Panao Pampa hamlets until reaching the Chicchuy village, Huanuco province (10◦00′13" S, 76◦12′17"W), Peru (see, Figure 1). Identification and authentication of the potato variety "walash" was used for elaborating tocosh and was carried out at the Natural History Museum of the Universidad Nacional Mayor de San Marcos (UNMSM), and a sample specimen was deposited with Ref. No. 038-USM-2020.

#### *2.2. Preparation of the Tocosh Flour*

The collected tocosh was washed in order to remove foreign matter and dust, then allowed to dry for three weeks under a shade in the place where it was obtained (−4 ◦C–18 ◦C). The dried tocosh was then pulverized using a grinder. The obtained product was named tocosh flour (TF) and stored until further use.

#### *2.3. Phytochemical Analysis*

A solution of tocosh dissolved and filtered was used to determine some phytochemicals such as alkaloids, phenols, terpenes, steroids, flavonoids, tannins, sugar, and saponins following the methodology of Herrera et al. [11]. The reaction to identify these components was done by using specific reagents for each chemical group showing any change of color or precipitation.

#### *2.4. Experimental Animals*

Balb/C albino mice and Holtzman rats of both sex were obtained from the bioterium of the National Institute of Health (Lima, Peru) with Sanitary Certificate No. 230-2019. Adult healthy male and female Holtzman albino rats (age, 12 weeks: body weight (males), 160–180 g; body weight (females), 150–170 g) were used to evaluate the sub-acute toxicity. Male and female rats were housed separately, and the selected female rats were nulliparous and non-pregnant. For acute toxicity, adult healthy male and female albino mice (age, 8 weeks: body weight (males), 30–32 g; body weight (females), 25–30 g) were used during the evaluation. These were kept in cages for approximately 15 days before the start of the study. The acclimatization of the experimental animals was carried out under environmental control conditions (12-h light/dark cycle) and temperature (22 ± 3 ◦C). Animals were given sterilized pellet food (National Center of Biological Products, NIH, Peru) and purified (reverse osmotic) water via a water bottle, ad libitum. All procedures were performed in reference to the institutional parameters and the guide for the care and use of laboratory animals. The protocol was presented and approved by the Ethics Committee of the Research Unit of the Faculty of Pharmacy and Biochemistry, UNMSM, (Document No 0198/FFB-UDI-2019. 11OCT2019. is certified with the REGISTRY No 010-CE-UDI-FFB-2).

#### *2.5. Sub-Acute Toxicity at Repeated Dose for 28 Days*

Sub-acute toxicity was performed in reference to the OECD 407 test guidelines [12]. Twenty rats were used and distributed in four groups. Group I (*n* = 5 males) and Group II (*n* = 5 females) named control groups, received only distilled water at repeated doses of 10 mL/Kg. Group III (*n* = 5 males) and IV (*n* = 5 females): rats received a limit dose of 1000 mg/Kg BW respectively, which according to animal safety criteria and empirical information for the consumption of tocosh by the population, which was established correspondingly to the repeated dose of 1000 mg/kg body weight, for 28 days. Each animal received a dose of tocosh flour suspension of 10 mL/kg body weight/day. The weights of each animal were recorded weekly during the treatment. After dosages of the product, the rats' body weights were measured and recorded during the test every 7 days until completion at 28 days.

On the 29th day, blood samples were collected by intracardiac puncture, under anesthesia with ethyl ether, biochemical and hematological parameters were evaluated. At the same time, all the animals were sacrificed with sodium pentobarbital (100 mg/kg) by subcutaneous route. The organs were fixed in 10% formalin for histopathology examinations.

Organs such as the heart, lungs, liver, spleen, stomach, brain, kidneys, and testes or uterus were removed immediately after sacrifice, washed with 0.9% sodium chloride, dried on filter paper, and weighed calculating the relative weights of the organs (ratio of organ weight and animal body weight (at the end of the experiment) × 100). Organs were examined for gross and/or microscopic pathology.

The biochemistry exams were performed with the Liquid Kinetic Chemistry method by using a clinical chemical analyzer brand MEDICA—EasyRA (5 Oak Park Dr, Bedford, MA 01730, USA), according to the manufacturer's specifications. The levels of aspartate aminotransferase (AST), alanine aminotransferase (ALT), alkaline phosphatase, total protein, bilirubin, total cholesterol, triglycerides, high-density lipoprotein (HDL), low-density lipoprotein (LDL), albumin, glucose, serum urea, and serum creatinine were determined.

Hematology examinations were performed by the flow cytometry method using a ZYBIO Brand Hematology Analyzer, Model Z31(Building J No. 70-1, 70-2 of Keyuan 4th Street Jiulongpo District, Chongqing Municipality 400039, China). The automated blood count (white blood cells, red blood cells, hematocrit, hemoglobin concentration, and platelet count) was evaluated.

#### *2.6. Acute Oral Toxicity—Fixed-Dose Study Procedure*

The acute oral toxicity of a fixed-dose procedure was evaluated according to the guidance of the Organization for Economic Cooperation and Development (OECD) method 420, with slight modifications in the animal selection, sex, and fasting [13]. This method grouped animals of both sexes dosing in a fixed-dose procedure using the highest doses of 2000 mg/kg and 5000 mg/kg (justified by criteria in animal welfare and related to the protection of human health based on the reference in the knowledge of empirical observation of the inhabitants of the area according to sample collection, being a product of frequent consumption in the area of the province of Huánuco).

Mice were kept with water ad libitum and were fasted for a fixed period such as 4–6 h before the administration of samples. Next, the animals were weighed and the test substance was administered. The toxicological evaluation was performed in 4 groups (*n* = 20). Group I (*n* = 5 males) and Group II (*n* = 5 females) received a single dose of 2000 mg/Kg body weight respectively, Group III (*n* = 5 males) and IV (*n* = 5 females): mice received a dose of 5000 mg/Kg BW, respectively. Each animal received a single dose of tocosh flour suspension at 10 mL/kg body weight. Mice were observed separately for 30 min, daily for 24 h, with rigorous observation in the first 4 h, and daily for 14 days. The individual weights of each mouse were determined before the administration of the test product and were re-calculated at the end of the 14 days.

The animal registry during the specific time of treatment was based on signs and symptoms of toxicity. The observations were recorded according to the duration of the treatment, including the specific external changes of minimal toxicity. At the end of the study, mice were sacrificed by sodium pentobarbital (100 mg/kg), immediately followed by necropsy. Organs were examined grossly for abnormal lesions.

#### *2.7. Histopathological Analysis*

Brain, heart, lung, liver, spleen. stomach, kidney, testes, and uterus of both studies (sub-acute and acute toxicity) were preserved in 10% formalin and fixed for a minimum of 24 h for a maximum of three days, dehydrated with alcohol of 70%, 96%, and absolute alcohol, the tissues were rinsed with xylol, impregnation with Paraffin, all these procedures were performed with a minimum of 1 h each. Inclusion in paraffin (formation of the paraffin block with the tissue) was performed, the cut was in a microtome, and at the last, they were sectioned at 3 µm depending on the tissue, and stained with hematoxylin and eosin (H&E). The sheets were assembled with coverslips and Entellan® (Sigma Chemical Co, St. Louis, MO, USA), which is a rapid non-aqueous mounting medium. Finally, the organ slides were examined microscopically and photographed with an optical microscope Nikon Eclipse E200 (Shinagawa Intercity Tower C, 2-15-3, Konan, Minato-ku, Tokyo 108-6290, Japan) at 40× and 200× magnification [14].

#### *2.8. Prediction of Drug-Likeness Properties for Steroidal Glycoalkaloids:* α*-Solanin,* α*-Chaconine and Solanidine*

Drug-likeness prediction along with further ADME properties presents wide of opportunities to evaluate a rapid prediction of chemical compounds with possible toxicological effects. The drug-like and ADME properties for the most active components of tocosh flour (constituents chemicals from *Solanum tuberosum* L., Figure 2) were screened using open-access cheminformatics platforms such as Molinspiration (for molecular weight-MW, rotatable bonds, and polar surface area-PSA descriptors), ALOGPS 2.1 (for Log Po/w descriptor) and the Pre-ADMET 2.0 to predicted four pharmaceutical relevant properties such as intestinal permeability (App. Caco-2), albumin-binding proteins (KHSA), Madin-Darby Canine Kidney (MDCK Line) cells permeation and intestinal absorption (%HIA). These parameters establish movement, permeability, absorption, and action of potential drugs [15]. The interpretation of both MDCK and Caco-2 permeability using PreADMET is as follows:


**Figure 2.** 2D-structures for the main alkaloids found in potato tubers [10].

#### *2.9. Statistical Analysis*

Data were recorded indicating a mean ± SD of five animals in each group and were analyzed by Student *t*-test in Graph Pad Prism software v6. The results were significant when the *p* value is less than 0.05.

#### **3. Results**

#### *3.1. Phytochemical Analysis*

The phytochemicals present in the solution of tocosh flour were sugars, phenols, alkaloids, saponins, and steroids.

#### *3.2. Repeated Dose Toxicity Study for 28 Days*

All female and male rats that received tocosh flour at doses of 1000 mg/kg/day for 28 days survived and no signs of toxicity were observed. In relative organ weights, livers of both sexes had a significant increase (*p* < 0.05), which was compared with the control group. The other organs evaluated did not have a significant difference (*p* > 0.05) compared to the control group (Table 1).

**Table 1.** Effect of tocosh flour solution on the relative organ weights in rats treated for 28 days.


Values are expressed as mean ± SD, compared with the control is significant when \* (*p* < 0.05). Data were analyzed using the Student *t*-test. No historical control values are available.

There were no cases of death for 28 days. Microscopic examination of each organ in female and male rats showed no abnormalities due to toxicity in any of the organs, such as liver, kidney, heart, lung, stomach, brain, spleen, testes, and uterus, compared to the control group (Figure S1).

At the end of 28 days, liver enzyme levels were maintained relative to the control group in male and female rats, there was no variation in the respective biochemical parameters, only a slight variation in the significant increase in LDL cholesterol was recorded in rats and decreased triglycerides, in male rats. (Table 2). In hematological parameter, there was no variation except for a slight significant variation of the percentage of monocytes in male rats, the other results of the hematological parameters did not have significant differences with the control group (Table 3).


**Table 2.** Biochemical parameters of rats after administration of repeated oral doses of tocosh flour at doses of 1000 mg/kg for 28 days.

Values are expressed as mean ± SD, compared with the control is significant when \* (*p* < 0.05). Data were analyzed using the Student test. AST (Aspartate aminotransferase); ALT (Alanine Aminotransferase); HDL (high density lipoprotein); LDL (low density lipoprotein). No historical control values are available.




**Table 3.** *Cont.*

Values are expressed as mean ± SD, compared with the control is significant when \* (*p* < 0.05). Data were analyzed using the Student test. No historical control values are available.

The body weight of the male rats administered with potato flour at a dose of 1000 mg/kg/day increased from the first week of 181.8 ± 9.28 g until the end of the fourth week, 261.4 ± 15.67 g, in contrast to the control group. In the control group of male rats, it was from 170.2 ± 9.20 g to 228.4 ± 20.24 g on day-28, which was significant (*p* < 0.05). Similarly, female rats administered at a dose of 1000 mg/kg had a weight gain that started in the first week at 172.8 ± 7.14 g to 228.4 ± 7.66 g corresponding to the end of the last week, compared to the control group of female rats which was significant (*p* < 0.001, see Figure 3).

**Figure 3.** Body weights of rats treated with repeated oral doses of tocosh flour (1000 mg/kg) for 28 days. (**a**) male rats and (**b**) female rats. \* *p* < 0.05, \*\* *p* < 0.001 compared to control, by using the Student *t*-test.

≤

### *3.3. Acute Oral Toxicity—Fixed Dose Procedure Study*

The individual weights of the mice to which the tocosh flour was administered at doses of 2000 and 5000 mg/kg were determined before the administration of the test product and were calculated and re-recorded at the end of the 14 days, with weight gain at the end of the experiment.

Mice administered at doses of 2000 mg/Kg and 5000 mg/kg body weight exhibited minor organ damage in the liver (parenchymal lymphocyte) and kidney (lymphocyte) according to Figures 4 and 5. However, no external aspects of toxicity were observed during the study linked to liver and kidney damage.

**Figure 4.** Liver tissue images of mice receiving a fixed dose of 2000 mg/kg and 5000 mg/kg of tocosh flour. (Stained with H&E, 200×). (**a**) Male group at the dose of 2000 mg/kg. Liver parenchyma with few lymphocytes around the central vein. (**b**) Female group at the 2000 mg/Kg dose. Liver parenchyma with isolated lobular lymphocytes (mild lobular inflammation). (**c**) Male group at the dose of 5000 mg/Kg. Liver parenchyma with isolated lobular lymphocytes (mild lobular inflammation). (**d**) Female group at the dose of 5000 mg/kg. Liver parenchyma with isolated lobular lymphocytes (mild lobular inflammation).

All this evaluation was followed in accordance with the OECD guideline 420, classifying the product as category B toxic (evident toxicity and/or ≤ 1 death), using the highest dose for safety and animal welfare to limit the number of animals, therefore the use of lower doses was restricted. In other organs, such as the brain, spleen, stomach, heart, and testes, no toxic damage was observed (Figures S2 and S3).

**Figure 5.** Renal tissue images of male and female mice that received a fixed dose of 2000 mg/kg BW and 5000 mg/kg BW of tocosh flour. (Stained with H&E, 200×). (**a**) Male group at the dose of 2000 mg/kg. Renal parenchyma with isolated chronic interstitial inflammatory infiltrate. (**b**) Female group at the dose of 2000 mg/kg. Renal parenchyma showing mild chronic interstitial inflammatory infiltrate with a tendency to lymphoid accumulation. (**c**) Male group at the dose of 5000 mg/kg. Renal parenchyma showing mild chronic interstitial inflammatory infiltrate with a tendency to the formation of lymphoid accumulation. (**d**) Female group at the dose of 5000 mg/kg. Renal parenchyma with isolated chronic interstitial inflammatory infiltrate.

#### *α α 3.4. Prediction of Drug-Likeness Properties for Steroidal Glycoalkaloids:* α*-Solanin,* α*-Chaconine and Solanidine*

α α We cannot show these results as toxicological finds due to these are only drug-likeness parameters. The properties showed the steroidal alkaloids as α-solanine, α-chaconine, and solanidine could be considered as pharmacokinetic behavior and were included because they are the main alkaloids found in *Solanum tuberosum* as is reported in other literature (Table 4).


**Table 4.**Calculated drug-likeness properties for glycoalkaloidsα-solanine,α-chaconine, and solanidine.

a Molecular weight of the hybrid (150–500). b Polar surface area (PSA) (7.0–200 Å<sup>2</sup>). c n-ON number of hydrogen bond acceptors ≤10. d n-OHNH number of hydrogens bonds donors ≤5. e Octanol water partition coefficient (log *<sup>P</sup>o*/*w*) (−2.0 to 6.5). f Binding-serum albumin (KHSA) (−1.5 to 1.5). g Human intestinal permeation (<25 poor, >500 great). h Madin-Darby canine kidney (MDCK) cells permeation.i Human intestinal absorption (% HIA) (>80% is high,<25% is poor).j Model for Human Oral Absorption.

#### **4. Discussion**

The glycoalkaloids from potatoes such as α-solanine, α-chaconine, and solanidine have a special function as a natural defense against plagues and its consumption may result in different symptoms that may include, diarrhea, fever, nausea, and even death [16,17]. The main studied mechanism of this kind of alkaloids is related to an inhibitory effect on the enzymes acetylcholinesterase (AChE) and butyrylcholinesterase (BuChE) [7,18]. Furthermore, it has been reported that glycoalkaloids interferes with ion-transport in cell membranes. The European Commission based on toxicological studies decided that the total glycoalkaloid content must not exceed the limit of 150 mg/Kg in potato protein powder for food applications [19]. However, the toxic effects produced by the inhibition of AchE and BuChE during the administration of tocosh flour were not observed in both subacute and acute toxicity treatments. Currently, there are no data on serious poisonings in the population, who consume tocosh flour as an alternative traditional treatment for digestive and respiratory diseases.

Tocosh comes from a fermentation process (Andean technique), which is suitable for distribution and consumption in the different markets of Peru as flour or in its raw form. On the other hand, the amount of glycoalkaloids are related to the cultivation method, storage and temperature, depending overall on the Andean techniques destined for its production and can be distributed in different rates in *Solanum tuberosum* tubers, they have been found in the tuber (smaller quantity), leaves and peel (greater quantity), and some analysis showed quantities such as 300–600 mg/kg in peel, 2000–4000 mg/kg in buds, and 3000–5000 mg/kg in flowers [20]. In our work, we did not evaluate the identification of constituents in tocosh flour by chromatography, but based on several studies of *Solanum tuberosum,* its glycoalkaloids are known by its fingerprint (α-solanine, α-chaconine and solanidine). Furthermore, probably other phytochemicals, lactobacillus and other could have been generated during the fermentation process and should be studied depending on the variety of potato and climatic factors. Otherwise, potato flour is an excellent food additive that can be applied in cakes, puffed food, breakfast food, baby food, condiments and soup and some functional factors such as anthocyanin, rosterone, and mucus proteins have been found, so its effect on health care is significant [21].

In regard to the repeated dose toxicity study using the limit dose of 1000 mg/Kg BW in rats, they had an increase in body weight. The increase of body weights differs from other studies where mice fed with 2130 mg/kg and 2170 mg/Kg of potato alkaloids such as α-chaconine and α-solanine for 7 days showed a decrease in body weight and in organs such as the liver, similarly in the administration for 14 days of the alkaloids: α-solasodine and solanidine [9,22]. This could be explained due to the animals being fed with tocosh flour which might contain a high content of carbohydrate and proteins, increasing the body weight in rats.

Significant increases in liver weight are commonly associated with adaptive changes such as accumulation of lipid, glycogen or other substances or a result of cell damage, congestion, hepatocellular hypertrophy or hyperplasia [23,24]. This variation does not always correlate with the amount of hepatic enzyme induction in rats as AST and ALT [25]. In our findings, we did not evidence any damage in liver tissues analyzed by using microscopy and any alteration in hepatic enzyme. This research revealed that presence of any phytochemical groups found in tocosh flour did not alter the histology in rats at the repeated dose of 1000 mg/kg BW for 28 days.

In hematological examination, there were no significant differences between rats administered with tocosh flour and the control group. The biochemical analysis LDL showed a significant increase in female and male rats. Similarly, there was a significant decrease of Tryglicerides in male rats but not in female rats. These findings might be altered due to potato starch, which is more phosphorylated than other starches of cereals [26], and indigestive polysaccharides promote excretion of bile acids, producing a reduction of Tryglicerides levels. However, an increase in LDL levels could be linked to carbohydrate consumption [27], thereby LDL production. No historical control data on the clinical chemistry or hematology values from the animal supplier were available for comparison; therefore, it is possible that the statistically changed parameters noted were within the normal range of average parameter values.

In acute toxicity according to OCED 420, minor changes in liver and kidney were observed with doses of 2000 and 5000 mg/Kg BW of tocosh administered in a single dose, probably attributed to its alkaloidal content and other components not determined in this study. *Solanum tuberosum* is known to have glycoalkaloids such as α-chaconin and α-solanine, mainly in tubers in almost 95%, also β-solanine, β-chaconin, γ-solanine, γ-chaconin, α-solamargin and β-solamargin but in less quantity [9,28]. However, these findings are unclear and not necessarily due to the consumption of tocosh flour.

The joint committee for food additives of FAO and WHO (JECFA) considers amounts of glycoalkaloids between 20 and 100 mg/kg as safe [29]. The toxic dose in the population corresponds to levels higher than 100 mg of total glycoalkaloids/kg of potato but this value could be influenced by environmental and storage conditions [17,30,31]. On the other hand, in the administration of 1.25 mg total glycoalkaloids/kg BW considered the highest dose in humans, gastrointestinal signs as vomiting appeared within 4 h [32,33]. Potato alkaloids at a dose of 75 mg/Kg of α-chaconin and α-solanin were lethally fatal within 4 to 5 days in Syrian golden hamster [34].

In an in vitro study, porcine oocytes were exposed to α-solanine (10 µM) and negatively affected early porcine embryo development by suppressing blastocyst formation and reducing embryo quality [35]. Another study found that the toxic and cytotoxic effects of α-Solanin in potatoes altered the proliferation and function of testicular cells in mice by regulating Sertoli and Leydig cells, affecting the testes and the reproductive function of male mice [36]. Likewise, α-solanine and α-chaconin at micromolar concentrations cause a cytotoxic effect on C6 rat glioma cells at the plasma membrane [37].

Currently, toxicological studies of the tocosh flour is not documented, according to the results obtained from in silico models, α-chaconine and α-solanine do not present Lipinski's rules, this also results in the permeability values calculated using the Caco-2 and MDCK models being so poor (<25 nm/s). Additionally, the oral and intestinal absorption values are very low (<25%). However, it is important to highlight that the prediction values obtained for its ability to bind to plasma transporter proteins are within the range of 95%, so they would have a high toxicity when it is given by intravenous administration. Nevertheless, solanidine showed results for a quick absorption and might be due to its small molecular weight and chemical structure not glycosylated. In animal studies, it has been shown that orally administered glycoalkaloids are less toxic than intraperitoneal administration (I.P.) due to poor absorption in the gut. In mice, LD<sup>50</sup> by I.P. administration have been reported to be 23 mg/Kg for α-chaconine, 34 mg/Kg for α-solanine, 500 mg/Kg for solanidine, and greater than 1000 mg/Kg for α-solanine by oral administration [38].

The NOAEL in rats of tocosh flour was 1000 mg/Kg, the estimate of the human equivalent dose (HED) [39] corresponds to a dose of 1000 mg as an initial dose in humans with 60 Kg of body weight. According to the traditional consumption of tocosh, this dose is less than the dose consumed by the population. Although, it seems to be safe compared with the limit dose of glycoalkaloids found in potato tubers.

In the present study, we could not identify and quantify each chemical constituent of tocosh potato. It is known that tocosh could contain other components such as lactobacillus and antibiotics produced during its fermentative process. However, tocosh can be consumed in established doses up to 1000 mg daily. Future studies of genotoxicity and chronic toxicity are needed as well to standardize its dose for consumption in foods.

#### **5. Conclusions**

The tocosh flour did not present toxicity at the repeated dose for 28 days in the highest dose corresponding to 1000 mg/kg BW. There were no deaths at up to 5000 mg/kg BW, therefore, the oral LD50 was greater than 5000 mg/kg.

**Supplementary Materials:** The following are available online at http://www.mdpi.com/2304-8158/9/6/719/s1, Figure S1: Images of some tissues of male and female mice without specific alterations that received fixed doses of 2000 mg/kg and 5000 mg/kg of Tocosh Flour stained with H&E and 40X, Figure S2: Images of the organ sections: Stomach (A), liver (B), Kidney (C) and heart (D), of male rats who were administered Tocosh flour for 28 days. Subscript C and 1 refer to the control group and the group administered at dose of 1000 mg/kg/day, respectively. H&E staining, 40X, Figure S3: Images of the organ sections such as: Stomach (A), liver (B), Kidney (C) and heart (D), of female rats that were administered Tocosh flour for 28 days. Subscript C and 1 refer to the control and group administered at dose of 1000 mg/kg/day, respectively. H&E staining, 40X.

**Author Contributions:** Conceptualization, O.H.-C. and J.R.V.-C.; methodology, J.R.V.-C., A.F.Y.-P. and J.P.R.-A.; software, C.A.; validation, R.D.H.-Q. and L.F.-S.; formal analysis, G.P.-R. and V.A.-A.; investigation, O.H.-C. and J.R.V.-C.; writing and editing-original draft preparation, O.H.-C.; writing—review and editing, O.H.-C., R.Á.Y.-P. and L.F.-S.; visualization, J.P.R.-A.; supervision, A.F.Y.-P. All authors have read and agree to the published version of the manuscript.

**Funding:** This research received no external funding.

**Data Availability:** The data sets used and/or analyzed during the current study are available from the corresponding author upon reasonable request.

**Conflicts of Interest:** The authors declare that there are no conflict of interest regarding the publication of this paper.

#### **References**


© 2020 by the authors. Licensee MDPI, Basel, Switzerland. This article is an open access article distributed under the terms and conditions of the Creative Commons Attribution (CC BY) license (http://creativecommons.org/licenses/by/4.0/).

*Review*

## **The Pharmacological Activity, Biochemical Properties, and Pharmacokinetics of the Major Natural Polyphenolic Flavonoid: Quercetin**

**Gaber El-Saber Batiha 1,2,\* ,**† **, Amany Magdy Beshbishy 1,**† **, Muhammad Ikram <sup>3</sup> , Zohair S. Mulla <sup>4</sup> , Mohamed E. Abd El-Hack <sup>5</sup> , Ayman E. Taha <sup>6</sup> , Abdelazeem M. Algammal <sup>7</sup> and Yaser Hosny Ali Elewa 8,9**


Received: 21 February 2020; Accepted: 20 March 2020; Published: 23 March 2020

**Abstract:** Flavonoids are a class of natural substances present in plants, fruits, vegetables, wine, bulbs, bark, stems, roots, and tea. Several attempts are being made to isolate such natural products, which are popular for their health benefits. Flavonoids are now seen as an essential component in a number of cosmetic, pharmaceutical, and medicinal formulations. Quercetin is the major polyphenolic flavonoid found in food products, including berries, apples, cauliflower, tea, cabbage, nuts, and onions that have traditionally been treated as anticancer and antiviral, and used for the treatment of allergic, metabolic, and inflammatory disorders, eye and cardiovascular diseases, and arthritis. Pharmacologically, quercetin has been examined against various microorganisms and parasites, including pathogenic bacteria, viruses, and *Plasmodium*, *Babesia,* and *Theileria* parasites. Additionally, it has shown beneficial effects against Alzheimer's disease (AD), and this activity is due to its inhibitory effect against acetylcholinesterase. It has also been documented to possess antioxidant, antifungal, anti-carcinogenic, hepatoprotective, and cytotoxic activity. Quercetin has been documented to accumulate in the lungs, liver, kidneys, and small intestines, with lower levels seen in the brain, heart, and spleen, and it is extracted through the renal, fecal, and respiratory systems. The current review examines the pharmacokinetics, as well as the toxic and biological activities of quercetin.

**Keywords:** quercetin; herbal remedies; pharmacological activities; pharmacokinetics; Alzheimer's disease

#### **1. Introduction**

Plants have been used since ancient times to cure certain infectious diseases, some of which are now standard treatments for several diseases [1,2]. Over the last decade, there has been a huge increase in acceptance and public interest in natural therapy in both developing and developed countries, and these herbal medicines are now available, not only in drug stores but also in supermarkets and food stores. Approximately 80 percent of people in Africa and other developing nations still depend on traditional herbal remedies to treat ailments due to their easy availability and lower cost compared to synthetic compounds [3,4]. They also demonstrate a number of promising activities against various health problems (e.g., respiratory and gastrointestinal disorders) and show anti-inflammatory, spasmolytic, antioxidant, sedative, antimicrobial, antiviral, antiseptic, anti-diabetic, immunostimulant, and hepatoprotective activities [5–7]. In addition, numerous phytoconstituents and plenty of chemical compounds with different biological and pharmacological activities have been isolated and identified from medicinal plants [8–10]. For instance, Batiha et al. [8], as well as Beshbishy et al. [9], reported the antiprotozoal activity of chalcones and ellagic acid, the naturally derived phytoconstituents isolated from herbal extracts against *Plasmodium*, *Leishmania*, *Trypanosoma*, *Babesia,* and *Theileria* parasites. These phytochemical compounds have been shown to be lead compounds for the development of new synthetic compounds, with higher efficacy and lower toxic side effects [11].

Quercetin (Figure 1: (2-(3,4-dihydroxyphenyl)-3,5,7-trihydroxy-4-Hchromen-4-one)) is classified as a flavonol, which is one of the six subcategories of flavonoid compounds and is the major polyphenolic flavonoid found in various vegetables and fruits, such as berries, lovage, capers, cilantro, dill, apples, and onions [12]. It is yellow in color and completely soluble in lipids and alcohol, insoluble in cold water, while sparingly soluble in hot water. Quercetin's name derives from the Latin word "*Quercetum*", which means Oak Forest, and also belongs to the flavonol category, which is not produced in the human body [13]. The name of the International Union of Pure and Applied Chemistry (IUPAC) and the chemical formula of quercitin are as follows: 2-(3,4-dihydroxyphenyl)-3,5,7-trihydroxychromen-4-one and C15H10O7, respectively. Quercetin is one of the most important plant molecules that has shown many pharmacological activities, such as being anticancer, antiviral, and treating allergic, metabolic, and inflammatory disorders, eye and cardiovascular diseases, and arthritis [14]. It has also shown a wide range of anticancer properties, and several reports indicate its efficacy as a cancer-preventing agent. Quercetin also has psychostimulant properties and has been documented to prevent platelet aggregation, capillary permeability, and lipid peroxidation, and enhance mitochondrial biogenesis [15]. The current review aims to further understand quercetin's beneficial and pharmacological effects, as well as its clinical application and concerns around safety.

**Figure 1.** Quercetin's chemical structure.

#### **2. Bioavailability and Pharmacokinetics of Quercetin**

Previous animal and human research have reported poor oral bioavailability of quercetin after a single oral dose due to macronutrient absorption [16]. For instance, quercetin is ingested in the form of glycosides, and glycosyl groups are released during chewing, digestion, and absorption. Afterward, quercetin glycosides are converted into aglycone in the intestine before they are absorbed into enterocytes by the action of β-glycosidases enzymes. According to Walle et al. [17], previous studies have reported that intestinal and oral bacteria are involved in this enzymatic hydrolysis. Quercetin is a lipophilic compound, so it is assumed that it can cross the intestinal membranes by simple diffusion, and theoretically, this absorption is better than its glycoside forms which reach the intestines without

degradation [18]. To date, a number of human studies have been conducted on the bioavailability of quercetin glycosides extracted from different species. For example, quercetin glycosides from onions were absorbed in patients with ileostomy at a higher percentage than pure aglycone, which has been reported by Hollman et al. [19]. On the other hand, Scholz and Williamson [20] documented the existence of significant amounts of aglycone in ileostomy fluid taken from patients with ileostomy who had eaten a meal with onions. They also reported the presence of a high quantity of quercetin glycosides and a small amount of quercetin aglycone, but quercetin glycosides were not observed in the fluid. One possible explanation is that the hydrolysis of quercetin glycosides takes place as it is converted by β-glycosidases enzymes to aglycone. These enzymes are found in the small intestine, and most of them are then absorbed. Ferry et al. [21] studied the pharmacokinetic properties of intravenous quercetin injection in cancer patients at dose levels of 60–2000 mg/m<sup>2</sup> . They determined that 945 mg/m<sup>2</sup> was a safe dose of quercetin, while its toxic dose was reported to cause emesis, hypertension, nephrotoxicity, and decreased serum potassium. The distribution and elimination half-life of intravenous quercetin were found to be 0.7–7.8 min and 3.8–86 min, respectively, whereas its clearance and distribution volume were 0.23–0.84 L/min/m<sup>2</sup> and 3.7 L/m<sup>2</sup> , respectively. Erlund et al. [22] examined the pharmacokinetic properties of 8, 20, and 500 mg quercetin aglycone orally in healthy participants. Graefe et al. [23] also studied the pharmacokinetic properties of quercetin and maintained a dose level of up to 200 mg and demonstrated that quercetin Cmax and Tmax were 2.3 ± 1.5 µg/mL and 0.7 ± 0.3 h, respectively.

#### **3. Sources of Quercetin and Its Pharmacological Activity**

Quercetin is one of the most significant bioflavonoid compounds found in vegetables, grains, and fruits for more than 20 plant species—namely, *Foeniculum vulgare*, *Curcuma domestica valeton*, *Santalum album*, *Cuscuta reflexa*, *Withania somnifera*, *Emblica o*ffi*cinalis*, *Mangifera indica*, *Daucus carota*, *Momordica charantia*, *Ocimum sanctum*, *Psoralea corylifolia*, *Swertia chirayita*, *Solanum nigrum*, and *Glycyrrhiza glabra, Morua alba*, *Camellia sinensis* [3], *Allium fistulosum*, *A. cepa*, *Calamus scipionum*, *Moringa oleifera*, *Centella asiatica*, *Hypericum hircinum*, *H. perforatum*, *Apium graveolens*, *Brassica oleracea* var. italica, *B. oleracea* var. sabellica, *Coriandrum sativum*, *Lactuca sativa*, *Nasturtium o*ffi*cinale*, *Asparagus o*ffi*cinalis*, *Capparis spinosa*, *Prunus domestica*, *P. avium*, *Malus domestica*, *Vaccinium oxycoccus*, and *Solanum Lycopersicum* [12]. It pharmacologically possesses antiobesity, anti-inflammatory, and vasodilator effects, and antioxidant, immunostimulant, anti-diabetic, antihypertensive, antiatherosclerosis, and antihypercholesterolemic activities (Figure 2) [24]. It is available as a food supplement in capsule and powder form.

**Figure 2.** The pharmacological activity of quercetin.

#### *3.1. General Pharmacological*/*Biochemical Properties of Quercetin*

Some of the sources and pharmacological activity of quercetin are shown below in Table 1.

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**Table 1.** Sources of quercetin and its traditional uses.

#### *3.2. Antioxidant Activity*

Interestingly, the beneficial effects of quercetin have been attributed to its antioxidant activity. Quercetin is a large class of flavonoids, consisting of five classes of hydroxyl groups, 3,5,7,3', and 4' of the basic flavonol skeleton. Some of these classes of hydroxyls are glycosylated to different quercetin glycosides and form the major quercetin derivatives. It is noteworthy that several studies have shown the relationship between the structural activities of quercetin and its derivatives on antioxidant and anti-inflammatory activities [25]. They found that the modification of quercetin reduces its antioxidant activity, and the total activity was found to be as follows: quercetin > tamarixetin = isorhamnetin > quercetin-3-O-glucuronide > isorhamnetin-3-O-glucoside > quercetin-3,5,7,3',4'-pentamethylether > quercetin-3,4'-di-glucoside, indicating that the 3-hydroxyl quercetin group plays a significant role in antioxidant activity [26]. Moreover, Lesjak et al. [25] reported that methylated quercetin metabolites (e.g., isorhamnetin and tamarixetin) showed higher antioxidant activity than quercetin by inhibiting lipid peroxidation. The antioxidant activity of quercetin has been documented because it can scavenge reactive oxygen species [27]. Quercetin is used to prevent cancer by modulating oxidative stress factors and antioxidant enzymes to prevent the spread of various cancers, such as lung, prostate, liver, breast, colon, and cervical cancers. The in vivo study examined the antioxidant activity of quercetin compared to carcinogen and testosterone by measuring histology and oxidative stress markers, such as reduced glutathione (GSH), lipid peroxidation (LPO), and hydrogen peroxide (H2O2) in rats. They found that rats treated with carcinogen and testosterone had higher levels of LPO and H2O<sup>2</sup> and lower levels of GSH compared to quercetin-treated rats [28]. Sharmila et al. [29] reported that quercetin increased the levels of apoptosis proteins and antioxidant enzymes in animals infected with prostate cancer. Moreover, they documented that quercetin regulated the expression of androgen receptors (AR), protein kinase B (AKT), insulin-like growth factor receptor 1 (IGFIR), and cell proliferation and anti-apoptotic proteins that are increased in cancer. In addition to that, quercetin has been documented to lower

malondialdehyde (MDA) content while increasing catalase and superoxide dismutase (SOD) activity to control the anti-inflammatory and anti-apoptosis processes to effectively protect the heart from secondary cardiac dysfunction due to oxidative stress and inflammation [30]. Quercetin also reduces the overproduction of ROS, damage caused by trauma, improves TNF-α, and prevents myocardial cell injury caused by Ca2<sup>+</sup> overload. Quercetin can thus effectively prevent injury caused by oxidative stress [31].

Interestingly, the antioxidant efficacy of quercetin has been documented in earlier reports to reduce and inhibit oxidative stress and damage, both in vivo and in vitro [32,33]. For illustration, Moretti et al. [34] demonstrated the efficacy of quercetin in the prevention of lipid peroxidation caused by *tert*-Butyl hydroperoxide in human sperm cells in vivo. An additional report in rats revealed that quercetin administered at dose levels of 25–50 mg/kg showed antioxidant action against oxidative stress, which results in streptozotocin-induced diabetes mellitus [35]. Moreover, it has been reported that quercetin acts as a stabilizer in the polyethylene when it is administered at a dose level of 250 µg/mL in addition to its antioxidant activity, and therefore the polymer's residual stability is increased for a long time [36]. Furthermore, the use of quercetin as a chelating agent in chelation therapy for the removal of toxic metallic ions such as cadmium as quercetin-cadmium complexes has been shown to have a high stability constant (Kf) value [37]. Quercetin reduces oxidative stress by controlling the oxidant–antioxidant balance. Several studies have reported that quercetin inhibits oxidative damage caused by acrylamide, brain damage caused by radiation in rats, neurodegenerative disorders, oxidative stress induced by cadmium fluoride, and nerve damage in diabetic rats' retinas. Quercetin protects the nerves, brain, or other body cells from oxidation-induced damage by regulating the antioxidant levels [31]. Quercetin prevents free radicals and enhances the body's antioxidant defense systems and therefore reduces oxidative stress, including the production of nicotine-induced ROS for the treatment of diseases such as nicotine addiction [38]. In vivo studies have shown that quercetin has antioxidant and hepatoprotective activity against acute hepatic injury caused by tertiary butyl hydrogen peroxide. Quercetin effectively protects cells from genetic toxicity and radiation-induced damage by scavenging free radicals and increasing the levels of endogenous antioxidants [39].

#### *3.3. Antiviral Activity*

Quercetin has shown antiviral activity towards a wide range of viruses. For instance, quercetin has been documented for its efficacy against the human T-lymphotropic virus 1, as well as the Japanese encephalitis virus (JEV) caused by Japanese encephalitis, the mosquito-borne disease [40,41]. Furthermore, quercetin has been reported to suppress the dengue virus type-2 and hepatitis C virus by suppressing the nonstructural protein 3 protease activity [42,43]. Other Quercetin formulations, such as quercetin-3-O-β-D-glucuronide, quercetin-enriched lecithin formulations, and quercetin 7-rhamnoside have been reported for their efficacy against the porcine epidemic diarrhea virus and influenza-A virus, respectively [44–46].

#### *3.4. Antimicrobial Activity*

Quercetin has exhibited potent bacteriostatic activity against different strains of bacteria, such as *Salmonella enterica* serotype Typhimurium, *Pseudomonas aeruginosa*, *P. fluorescens*, *Helicobacter pylori*, *Staphylococcus epidermidis, S. aureus*, *Yersinia enterocolitica*, *Micrococcus luteus, Campylobacter jejuni,* and *Escherichia coli,* which have been more effective against Gram-positive than Gram-negative bacteria [47]. Jaisinghani. [48] also reported its efficacy against *Shigella flexeneri* NCIM5265 and *Lactobacillus casei var Shirota.* Strikingly, Osonga et al. [49] documented that quercetin derivatives (e.g., quercetin 4 ′ ,5-diphosphate (QDP), quercetin 3′ ,4′ ,3,5,7-pentaphosphate (QPP), quercetin 5′ -sulfonic acid (QSA)) resulted in highly biocompatible, soluble, and potent antibacterial activity with 100% inhibition of *Listeria monocytogenes*, *Pseudomonas aeruginosa*, and *Aeromonas hydrophila*. Moreover, quercetin revealed the strongest antifungal activities against *Candida albicans, Cryptococcus neoformans,* and *Aspergillus niger* [50].

#### *3.5. Antiprotozoal Activity*

Several reports have demonstrated the growth inhibitory effects of quercetin against various protozoan parasites, namely *Toxoplasma*, *Babesia*, *Theileria*, *Trypanosoma,* and *Leishmania*. Interestingly, quercetin is well-known for its growth inhibitory efficacy against *Trypanosoma brucei rhodesiense, T. brucei brucei, T. cruzi,* and *Leishmania donovani* parasites in vitro and in vivo [51]. It resulted in potent leishmanicidal and trypanocidal activity in vitro, with an IC<sup>50</sup> of 1.0 µg/mL and 8.3 µg/mL, respectively, while in an in vivo experiment, among six tested flavonoids, only quercetin showed in vivo activity by inhibiting the multiplication of *L. donovani.* Moreover, Weiss et al. [52] documented the remarkable inhibitory effects of quercetin against *Toxoplasma gondii* by preventing the heat shock protein 90 (hsp90), hsp70, and hsp27 synthesis, and thus suppressing the induction of bradyzoite development. Lehane and Saliba. [53] described the antiplasmodial activity of quercetin against a chloroquine-sensitive (3D7) and chloroquine-resistant (7G8) strain of *Plasmodium falciparum.*

#### *3.6. Anti-Inflammatory E*ff*ects of Quercetin*

Quercetin has been shown to be a long-lasting anti-inflammatory agent with good anti-inflammatory activity. Several in vitro studies have shown that quercetin prevents the development of lipopolysaccharide (LPS)-mediated tumor necrosis factor-α (TNF-α) in macrophages and the development of IL-8 induced LPS in lung A549 cells [54]. In addition, quercetin can inhibit TNF-α and Interleukin (IL)-1α levels of LPS-induced mRNA, which results in reduced apoptotic neuronal cell death caused by microglial activation [55]. Quercetin suppresses the production of inflammatory enzymes (e.g., lipoxygenase (LOX) and cyclooxygenase (COX)). It regulates inflammation caused by LPS by inhibiting Src- and Syk-mediated phosphatidylinositol-3-Kinase (PI3K)-(p85) tyrosine phosphorylation and subsequent complex formation of Toll-like Receptor 4 (TLR4)/MyD88/PI3 K, which restricts downstream signaling pathway activation in RAW 264.7 cells [56]. It may also inhibit the release of pro-inflammatory cytokines, tryptase, and histamine from human umbilical cord blood-derived mast cells; this inhibition is likely to involve the inhibition of calcium influx and Phospho-protein kinase C (PKC) [54]. Quercetin substantially stimulates the gene expression and the development of interferon-γ (IFN-γ) derived from T helper cell-1 (Th-1) and down-regulates IL-4 derived from Th-2 by normal peripheral blood mononuclear cells (PBMC). Quercetin is also known to have inhibitory activity against COX-2, nuclear factor-kappa B (NF-κB), activator protein 1 (AP-1), mitogen-activated protein kinase (MAPK), reactive nitric oxide synthase, (NOS) and reactive C-protein (CRP) expression that causes inflammation [57]. Due to its weak absorption through the surface of the skin, quercetin and its glycoside derivatives have been reported to be ineffective against topical inflammation, while pentamethyl ether, which is a quercetin derivative, has shown potent anti-inflammatory activity with higher absorption through the skin's surface in the rat [58]. Several reports have been documented that quercetin prevents the secretion of iNOS, IL-1β, and TNF-α caused by bacterial LPS in macrophages, TNF-α secretion in RAW2647 cells, and cytokine-stimulated vascular cell adhesion molecules (VCAM-1) and intracellular cell adhesion molecule (ICAM-1) expression, and E-selection in human umbilical vein endothelial cells. Notably, quercetin and its glycoside rutin have shown a reduction in the inflammatory markers TNF-α and IL-6 in NASH mice [59].

#### *3.7. E*ffi*cacy in Diseases*

#### 3.7.1. Anticancer Activity of Quercetin

Quercetin has been documented to possess anticancer activity both in vitro and in vivo. In in vitro experiments, the anticancer efficacy of quercetin against different cell lines was determined by the prevention of angiogenesis in tamoxifen-resistant cancer, while its in vivo efficacy was attributed to its antioxidant activity [60–62]. According to Gibellini et al. [63], quercetin is considered to be a strong anticancer candidate due to its chemoprotective activity through metastasis and apoptosis against tumor cell lines. Moreover, Du et al. [64] demonstrated the potent efficacy of the quercetin-doxorubicin combined treatment in persistent T-cell tumor-specific responses, resulting in improved the immune responses against breast tumor growth [64]. It is worth noting that quercetin prevents the proliferation of several types of cancers (e.g., breast, lung, prostate, cervical, liver, and colon cancer) and it acts by a various mechanism of actions, including cellular signaling, binding to cellular receptors and proteins, and inhibiting enzymes responsible for carcinogen activation [65]. Recently, quercetin has been reported to increase the chemosensitivity of breast cancer cells to doxorubicin by preventing cell propagation and invasion that promote cell apoptosis. Furthermore, quercetin demonstrated an inhibitory effect on MCF-7 and MDA-MB-231 human breast cancer cell lines by regulating miR-146a expression, cell apoptosis induction, Caspase 3 activation, and mitochondrial pathways [66]. Quercetin also exhibits anti-colon cancer effects with the TLR4- and NF-κB-mediated signaling pathway, and it was found that quercetin showed significant inhibition of human colon cancer proliferation in CACO-2 and SW-620 cells by preventing the NF-κB pathway, down-regulation of B-cell lymphoma 2, and up-regulation of Bcl-2-associated X protein [67].

#### 3.7.2. Quercitin Hepatoprotective and Antihypertensive Activities

Recently, an in vivo study found that quercetin increased heme oxygenase 1 activity in D-galactosamine- and LPS-treated rats by lowering plasma concentrations of alanine aminotransferase and stimulating its hepatotoxic and hepatoprotective activity [68]. Moreover, Liu et al. [69] revealed the ability of quercetin to treat ethanol-induced oxidative damage in rat hepatocytes, suggesting that quercetin may be an appropriate hepatoprotective natural product. Duarte et al. [70] reported that quercetin had antihypertensive activity in spontaneously hypertensive rats, and noted that quercetin had induced a dose-dependent, advanced, and potential reduction in pressure of the blood when given chronically to several hypertensive rat models.

#### 3.7.3. The Important Role of Quercetin in the Treatment of Alzheimer's Disease

Alzheimer's disease (AD) is considered to be the most prevalent cause of dementia, a chronic neurodegenerative disorder characterized by memory loss and mental deficits, such as apraxia, aphasia, and agnosia, and is associated with neuroinflammatory processes in the central nervous system [71,72]. The memory contains several types: visual, olfactory, episodic, and vocal. These are classified into two categories: explicit (active or passive recall of facts) and implicit (nonverbal habitual memory) [73].

Oxidative stress is caused by a free radical imbalance in the body and is included in the establishment of neurodegenerative disorders involving AD. Flavonoids like quercetin have different activities in the vascular system, leading to several modifications in cerebrovascular blood flow, which can alter the neuronal morphology that causes neurogenesis and angiogenesis. In addition to that, it can also protect neurons from neurotoxin-induced injury. Rich food consumption of flavonoids limits neurodegeneration and inverts the age-related injury to cognitive performance [74]. Moreover, quercetin and ascorbic acid combined treatment have been shown to reduce the prevalence of oxidative injury to human lymphocytes and neurovascular structures in the skin and thus prevent neuron injury, which particularly protects the brain cells from oxidative stress that leads to AD and other neurological conditions [13].

Quercetin's beneficial effects against AD are ascribed due to its inhibitory efficacy against acetylcholinesterase (AChE) [72]. Recently, in vivo experiments have documented the ability of quercetin to reduce the oxidative stress caused by 6-hydroxydopamine in the neurons of rats [75]. Another study conducted on healthy P19 neurons revealed that neuron survival is not affected by quercetin, while it depletes the glutathione content that may affect the functioning of the nervous system [76]. Furthermore, recent findings have shown that quercetin improves the pathology of AD and related cognitive deficits in triple-transgenic, aged AD mice [77]. Additionally, combined oral ingestion of quercetin with fish oil improved neuroprotection in 3-nitropropionic acid-treated rats or chronic rotenone-treated rats [78,79].

In the AD, quercetin acts by the following mechanism of action: α-tocopherol (vitamin E), a type of antioxidant that enhances quercetin penetration through the blood–brain barrier (BBB), which leads to significant improvement in quercetin concentration and thus reduces the prevalence of oxidative damage in the brain. Moreover, quercetin acts by activating the NF-E2-related factor 2- antioxidant responsive element (Nrf2-ARE) that offers a neuroprotective effect against oxidative injury and cell death. Recently, previous studies have shown that the formation and deterioration of undisciplined protein aggregates in various neurodegenerative diseases, such as Huntington's diseases, Alzheimer's, Parkinson's, and amyotrophic lateral sclerosis may be altered by the Nrf2-ARE pathway [80,81].

#### **4. Combination Therapy of Quercetin with Other Drugs**

The combined effect of quercetin with other antioxidants (e.g., ascorbic acid), decreases the prevalence of oxidative damage in human lymphocytes and neurovascular structures in the skin and inhibits the neuron injury. Moreover, it has been reported to possess a potent effect against AD by protecting the brain cells from oxidative stress that induces tissue damage, resulting in AD and other neurological conditions [13]. Notably, quercetin has been documented to possess neuroprotective and neurotoxic activity, and its combined effect with fish oil has shown neuroprotective efficacy in rat brains and has subsequently shown beneficial effects against neurodegenerative diseases [79]. Quercetin is well-known to influence the pharmacokinetics of different drugs, such as curcumin and resveratrol by controlling their transfer and metabolism, as well as some of its significant activities, including CYP3A4, P-gp efflux pump, and phenol sulfotransferase (SULT 1A1) inhibition. These combined treatments have resulted in an increase in curcumin and resveratrol permeability and acute bioavailability compared to single treatments [82]. Moreover, Sahyon et al. [83] investigated the combination effect of sulfamethoxazole with quercetin against *S. aureus*, and quercetin has been shown to reduce the side effects of sulfamethoxazole while improving its bactericidal efficacy, indicating the importance of this combination therapy for the treatment of human clinical cases. Also, Qu et al. [84] revealed the synergetic effect of quercetin-tetracycline combination treatment against multi-drug resistant (MDR) *E. coli* by disrupting the bacterial cell envelope, thus improving its permeability and cell lysis. Quercetin has been documented to improve the antifungal efficacy of amphotericin B against *Candida* sp and *Cryptococcus neoformans* strains by reducing its toxic effect [85]. Another study demonstrated the potent synergistic efficacy of quercetin against fluconazole-resistant strains of *Candida tropicalis* by enhancing mitochondrial membrane alterations that affect the mitochondrial respiratory function and inhibiting rhodamine-123 accumulation in the mitochondria [86]. Recently, quercetin has been documented to possess synergistic effects when combined with chemotherapeutic drugs (e.g., cisplatin) [65].

#### **5. Dose Use**

Typical dietary quercetin intake based on fruit and vegetable consumption is estimated to range from 5 to 100 mg per day. Heavy consumption of foods rich in quercetin, such as apples or onions, could lead to a daily intake of up to 500 mg [87–89]. The effective dose is increased when taken with a fatty meal or in the presence of apple pectin, oligosaccharides, and lecithin [87,90]. Most clinical studies use quercetin at 500 to 1000 mg per day in divided doses [91,92]. As a supplementary food, 2 weeks of quercetin 50 mg achieved a 178% increase in serum levels, while quercetin 100 mg had a 359% increase in the serum levels, and quercetin 500 mg had a 570% increase in the serum levels, although with wide individual variation [93]. Based on animal studies, quercetin accumulates in the lungs, liver, kidneys, and small intestines, with lower levels seen in the brain, heart, and spleen. It is eliminated through the renal, fecal, and respiratory systems [89,94].

#### **6. Metabolism and Excretion of Quercetin**

After quercetin administration and absorption, it is transferred to the liver where the first and second phases of metabolism take place, resulting in metabolic products entering the bloodstream for distribution in the body's tissues [95]. Mullen et al. [96] examined the main metabolites of quercetin in the urine and plasma of healthy people after the ingestion of onions. Three major metabolites were identified in the plasma—namely, quercetin-30-sulfate, quercetin-3-glucuronide, and quercetin-3-sulfate with the highest concentrations at 0.8 and 0.6 h, while quercetin-30-glucuronide, quercetin-diglucuronide, isorhamnetin–glucuronide sulfate, isorhamnetin-methyl quercetin, and diglucuronide isorhamnetin-glucuronide were the major urinary metabolites that reached their highest concentrations after 4 h. Notably, quercetin had a short half-life and rapid clearance in the blood, and its metabolites appeared in the plasma 30 min after ingestion, but considerable amounts were excreted over 24 h [97]. Moon et al. [94] identified the aggregation of quercetin conjugates in human plasma following multiple administrations of quercetin-rich foods. The highest concentration of quercetin metabolites was identified following the uptake of onions, and sulfate and glucuronide metabolites were significantly (*p* < 0.05) elevated from 0.04 to 0.63 µM in the plasma of fasting participants.

The use of quercetin in the pharmaceutical industry is limited due to its poor bioavailability, poor aqueous solubility, poor permeability, and instability. Therefore, several studies have been conducted to modify its structure to increase its water solubility and bioavailability and thereby enhance its antioxidant and antimicrobial activity [98]. Recently, new quercetin preparations have appeared, including quercetin-loaded gel, quercetin-loaded mucoadhesive nanoemulsion, quercetin-loaded nanoparticles, and quercetin-loaded polymeric micelle, which may provide new drug formulations for research and development (Figure 3) [31]. Moreover, quercetin bioavailability has also been improved by structural modification with glucoside–sulfate conjugates and the preparation of some complex ionic complexes, such as quercetin–germanium nanoparticles, calcium phosphate–quercetin nanocomplex (CPQN), and glucan–quercetin conjugate that showed higher antioxidant activity than free quercetin [99]. Quercetin also exhibits excellent antioxidant activity and scavenging capacity when combined with metal ions, such as cadmium, vanadium, calcium, magnesium, copper, cobalt, iron, and ruthenium [31].

**Figure 3.** Quercetin formulations for improving its bioavailability.

#### **7. Toxic Side E**ff**ects of Quercetin**

Quercetin is known to be a mutagenic agent based on the Ames test; however, most in vivo animal studies have shown that quercetin is a safe compound without any carcinogenic effects. It is worth noting that in 1999, the International Agency for Research on Cancer (IARC) stated that quercetin should not be listed as a human carcinogen compound [100,101]. There is no definite proof of quercetin teratogenic activity on embryonic growth; however, in vitro studies suggest that quercetin can have a mild negative impact on fetal growth and demonstrate protective efficacy against toxic agents [102]. In vivo experiments have shown that quercetin resulted in a small increase in the prevalence of malignant tumors to the young offspring of mice lacking DNA repair [103]. An in vivo experiment performed on a four-week rat showed that the ratio of liver and kidney weights increased remarkably in rats fed greater than 314 mg and 157 mg quercetin/kg body weight/day, respectively. Moreover, a pro-oxidant efficacy was observed at doses higher than 157 mg quercetin/kg body weight/day [104]. Quercetin was usually well-tolerated in human clinical studies. Notably, quercetin administration for several months at a concentration higher than 1000 mg/day did not show any side effects on serum electrolytes, kidney, and liver function blood parameters, or hematology. At present, co-administration of high quercetin doses with digoxin is known to be the greatest cause of toxicity; thus, the use of quercetin in digoxin-treated patients should be restricted before more information on appropriate dosage levels is available [105]. Quercetin shows mutagenicity in vitro in the Ames test, and reports of mutagenicity in the 1970s have led to concerns about its safety [87]. Under certain circumstances, quercetin exhibits both radical scavenging and pro-oxidant activity [88].

The majority of in vivo experiments have shown that quercetin is not a carcinogen and may be protective against Geno toxicants. Dietary quercetin, faced with the first-pass metabolism in the intestine and liver, is almost completely metabolized, reducing the potential for toxicity. At oral supplemental doses higher than 1000 mg per day taken for up to three months, no evidence of toxicity has been found; however, data on long-term safety at high doses are lacking [87]. Nephrotoxicity has been reported with the use of high-dose IV quercetin in patients with compromised health [89].

#### **8. Quercetin-Drug Interaction**

Quercetin has been reported to competitively bind to bacterial DNA gyrase and is, therefore, contraindicated to be administered with fluoroquinolone antibiotics [106]. Moreover, quercetin is a potent competitive inhibitor of CYP3A4 (the enzyme responsible for drug degradation in the body) and was thus predicted to increase the serum concentrations of drugs (e.g., diltiazem) that are metabolized by this enzyme [107]. Therefore, research towards the optimum mechanism of action of natural compounds that prevents the adverse effects of plants and the development of new molecules with new pharmacological effects will continue [108–110].

#### **9. Conclusions**

This review examined the therapeutic and toxic activities of quercetin. Quercetin is the major polyphenolic flavonoid present in several food products that have shown many pharmacological activities, such as anticancer, antiviral, antiprotozoal, and antimicrobial effects, treatment of allergic, metabolic, and inflammatory disorders, eye and cardiovascular diseases, and arthritis. Previous studies documented the poor oral bioavailability of quercetin after a single oral dose, as its absorption was impaired by the macronutrients. It is a famous AChE inhibitor and has been used in treating neurodegenerative diseases, including AD. Quercetin has been documented to have both neurotoxic and neuroprotective activities, and its combined effect with fish oil and ascorbic acid has demonstrated beneficial effects against neurodegenerative diseases. The finding that quercetin can combine with other drugs is a property that can be explored in the chemotherapies' development against AD. However, due to the presence of adverse side effects, its therapeutic use as a treatment has been banned. Moreover, quercetin has been confirmed to be competitively bound to bacterial DNA gyrase and is, therefore, contraindicated to be administered with fluoroquinolone antibiotics.

**Author Contributions:** A.M.B., G.E.-S.B., Z.S.M., M.I., M.E.A.E.-H., A.E.T., A.M.A., and Y.H.A.E. wrote the paper. A.M.B. and G.E.B. revised the paper. All authors have read and agreed to the published version of the manuscript.

**Funding:** This research received no external funding.

**Conflicts of Interest:** The authors declare no conflict of interest.

### **Abbreviations**


### **References**


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