Effects of Glycyrrhiza Glabra (Licorice) Root Extract on the Hormones, Serum Biochemicals, and Hematological Parameters in Dogs with Benign Prostatic Hyperplasia

Benign prostatic hyperplasia (BPH) is the most common prostate disorder in non-neutered dogs and is often caused by androgenic stimulation or changes in the ratio of androgen to estrogen. Also, it is commonly seen in neutered older dogs. Clinical signs may not be presented, but persistent or intermittent hematuria, hemospermia, or a primary hemorrhagic discharge are usually reported. In this research, ethanolic extract of licorice root (EELR) was used as the treatment, which has various antioxidant and healing properties and can reduce lesions by establishing an antioxidant balance and reducing inflammation. For this experiment, 30 dogs of approximately the same age and weight were randomly divided into 6 groups of 5 each. The treatment groups received the EELR at doses of 10 and 20 mg/kg separately, while the control group received no medications. At the end of the 9-week treatment period, biochemical and hormonal factors were measured and analyzed by blood sampling. The results showed that the EELR has multiple positive effects on the serum biochemical indices and also positively affects hormone levels, while it can decrease the prostate-specific antigen (PSA) level in BPH dogs. In conclusion, the EELR can effectively reduce BPH-induced lesions without any side effects.


Introduction
Te prostate is a gland behind the bladder that is located in the pelvis, directly below the rectum. Prostate disease is most common in humans, chimpanzees, and dogs, and rarely in cats. Te disease is most common in 80% of neutered dogs over 5 years of age and 95% of neutered dogs over 9 years of age [1].
BPH is the most common prostate disease in nonneutered dogs [2]. As expected, no breed predisposition for BPH has been observed, and it may occur in almost any intact male dog [3]. One of the most important aspects in cases of BPH-related complications is to examine their correlation with urinary tract diseases, especially the diseases of the lower urinary tract, urinary tract infections, and kidney failure. Unlike prostatomegaly, which occurred in many cases, some of the dogs showed no clinical signs [4], so the protocol for BPH detection should be revised. Since surgery is not possible in many cases, medication therapy can be the choice of many patients to treat or alleviate the disease, among which is herbal medicine due to its natural antioxidants that can have a high medicinal potential [5]. Tis is an important issue because, generally, BPH damage can afect the healthy condition of other organs, such as the kidneys, and alter markers such as the creatinine and serum urea levels in the blood, both of which indicate the kidney's health [6].
Licorice has many active ingredients, which have shown some promising potentials, such as Glycyrrhizic acid, which has shown high anti-infammatory, anticancer, and antioxidant potentials [7][8][9]. Te leaf and root parts of this plant are rich in natural active ingredients, and the extracts of these parts of the plant are very rich in these active ingredients [10]. Tis plant also contains a high level of triterpenes, saponins, favonoids, and several phenolic acids [11], which are proven to have antioxidant and anticancer efects [12,13]. Glyceric acid and glycerin are useful for treating gastrointestinal ulcers [14]. Te roots of the licorice plant also contain coumarin, favonoids, essential oils, and plant sterols. In traditional medicine, this plant is used to treat muscle spasms and swelling, bronchitis, rheumatism, and arthritis [15].
One of the important points in cases of benign prostatic hyperplasia is the correlation of their primary disease with secondary urinary tract diseases, especially the lesions of the lower urinary tract. Urinary tract infections and kidney failure are only a small part of the pet owners' complaints when they visit a clinic. Since surgery is not possible in many cases, the medication for treating or alleviating the disease would be the frst choice of many patients [5]. Regarding the association of BPH with renal problems, monitoring the renal health indices would be helpful [16,17].
In this research, the efects of EELR on biochemical renal indices were investigated. Also, BPH following oxidative stress and hormonal misregulation were examined.

Study Design and Animal
Grouping. BPH-suspected male dogs were physically examined, and a prostate test was performed to assess the parenchymal uniformity and the presence or absence of a cyst [18]. Ten the susceptible dogs were identifed due to factors such as nonuniformity, heterogeneous parenchyma, or the presence of cysts, which all greatly increase the risk of BPH. Finally, to ensure that the dogs were BPH-positive, an ultrasound was used, the prostate volume was examined, and BPH of the dogs was confrmed [19]. After fnding 15 dogs with the BPH and 15 healthy dogs, which all had the same body condition (age and weight), they were randomly divided into 6 groups of 5 each. All dogs were kept under the same conditions. Tey all shared the same food and water. For the treatment, the ethanolic extract of licorice root (EELR) was administered orally to the dogs 3 days a week for 9 weeks. Finally, after 9 weeks, blood samples were taken from all the dogs from the cephalic vein. All dogs survived until the last day of the project. Male dogs were randomly divided into 6 groups of 5 each, including the control group (healthy dogs), the BPH group (sick dogs), the EELR group at a dose of 10 mg/kg (healthy dogs), the EELR group at a dose of 20 mg/kg (healthy dogs), the frst treatment group of the EELR with the dose of 10 mg/kg (sick dogs), and the last treatment group of the EELR at a dose of 20 mg/kg (sick dogs) [20]. In the control and BPH groups, the dogs were fed water and food and did not receive any chemicals or medication. Te ethical protocol of ARRIVE [21] was followed throughout the whole project, and the research ethics committees of the Islamic Azad University approved the project with the approval ID of IR.IAU.BABOL.REC.1400.083.

Extract
Preparation. 2 kg of freshly dried licorice root were purchased and powdered. Next, it was extracted by solving it in absolute ethanol, and then the extract was stored in a refrigerator at 4°Celsius until it was used [22].

Gas Chromatography Analysis of EELR.
After extraction, 2 ml of the ethanolic extract of licorice root (EELR) was placed in a gas chromatography machine (Shimadzu GCMS-TQ8040 NX) and analyzed. Te reported substances were specifed by comparing their spectra with those in Wiley and the NIST/EPA/NIH34-44 spectral mass libraries [23,24].

Blood and Serum Tests.
All the blood and serum markers were assessed by the autoanalyzer. Te hormones and stress markers were also estimated with special kits.

Statistical
Analysis. Blood factors were analyzed using SPSS software version 26 and a one-way ANOVA with a Tukey post hoc test. Te minimum signifcance level was set to P < 0.05 [22].

GC-MS of EELR.
Te ethanolic extract of licorice root was placed inside a gas chromatographic machine, and the results were read after 3 repetitions. Components that were less than 1% were removed from the table and merged as Others (Table 1).

Complete Blood Count (CBC).
Te number of red blood cells (RBC) in the BPH group was higher than that of the control group, while in the BPH + EELR groups, a slight decrease was observed compared to the BPH group. Te hemoglobin (HGB) was not signifcantly diferent between the groups, but it should be noted that its number went down in the EELR groups compared to the control. Te hematocrit (HCT) in the BPH group was higher than that of the control group. Te levels of other factors were also elevated in the BPH group compared to the control group, but no signifcant diference was seen ( Table 2).
Platelet levels were increased in the BPH groups compared to the control group, which then decreased in the BPH + EELR10 group. Also, in the EELR groups, the EELR20 showed a moderate increase in platelet levels compared to the control group. PCT levels were not signifcantly diferent in all groups. Te level of MPV in the BPH group showed an increase compared to the control group, while in all groups that were afected by the EELR, we saw a clear fall compared to the control and BPH groups. PDW levels did not difer signifcantly between the groups (Table 3).

White Blood Cell Count (WBC).
Te WBC count in the BPH group was lower compared to the control group. However, in all groups that were afected by the EELR, we saw a moderate increase in the WBC compared to both the control and BPH groups. Tere was no signifcant diference in the neutrophil percentages between all groups. On the other hand, slight increases in the percentages of lymphocytes, monocytes, and eosinophils were observed in the BPH group compared to the control group, which were also not signifcant (Table 4).

Serum Biochemical Parameters.
Te levels of both albumin and total protein (TP) indices were slightly increased in the BPH group compared to the control group, but no signifcant change was observed. Regarding insulin, we saw a decrease in its level in the BPH group compared to the control group, which was even lower in the EELRreceiving groups, which again was not signifcant. Consequently, glucose levels did not show a signifcant difference between the groups, but a slight increase in the glucose level of the BPH + EELR10 group was observed. ALT and AST levels were increased in the BPH group compared to the control group, while they were decreased in the BPH + EELR groups compared to the BPH group moderately (Table 5).

Kidney Parameters.
Te blood urea nitrogen (BUN) level in the BPH group was increased compared to the control group, but in the BPH + EELR20 group, it was substantially reduced. Tere was no signifcant diference in the uric acid levels between the groups. Te creatinine level was also increased in the BPH group compared to the control group, while it was decreased in both treatment groups compared to the BPH group. Te amounts of calcium and phosphorus in the groups were not signifcantly different from each other ( Table 6).

General Cell Damage
Indices. Levels of both LDH and CPK indices were increased in the BPH group compared to the control group. Meanwhile, a decrease in both of these markers was seen in the treatment groups, which was more in the BPH + EELR20 group (Table 7).

Hormones and PSA.
Te PSA level in the BPH control group was signifcantly higher than the control group (P < 0.0001). In the BPH + EELR20 group, a signifcant fall in   Evidence-Based Complementary and Alternative Medicine the PSA level was observed compared to the BPH group (P < 0.01). However, there was not a signifcant diference between the EELR10 and the BPH group. Also, no signifcant diference was observed between the EELR-only and control groups. Te estradiol and testosterone levels showed a signifcant decrease (P < 0.0001) in the BPH group compared to the control group. In both cases, the EELR was able to increase the levels of these hormones, with the EELR20 increasing both signifcantly (P < 0.0001) and bringing them closer to those of the control group (Figure 1).

Oxidative Stress Indices.
After examining the levels of serum stress markers, it was observed that the level of malonaldehyde (MDA) in the dogs with BPH was signifcantly higher than that of the control group. In the EELR10 treatment group, it was seen that the MDA level was signifcantly decreased (P < 0.05) compared to the BPH group, and in the EELR20 treatment group, this increase was even more (P < 0.0001). Te total antioxidant capacity (TAC) level in the BPH group was signifcantly (P < 0.05) lower than the control group, but signifcantly higher in the BPH + EELR20 group compared to the BPH group (P < 0.01) (Figure 2).

Discussion
In this study, the efect of ethanolic extract of licorice root (EELR) at the two doses of 10 mg/kg and 20 mg/kg on dogs with BPH was investigated, and in addition, the toxicity of these two doses was examined separately. Te licorice extract     [25]. Te efects of the diferent natural substances were previously studied on BPH human patients [26,27], and it was shown that they could be efective. Even a positive anticancer efect was seen in an earlier study of the licorice extract [28]. Initially, after the GC-MS result of the EELR, it was observed that this extract has many favonoids and antioxidants, each of which has the potential to decrease infammation and tissue damage such as hyperplasia [29]. It was shown that the upper part of this plant has many benefcial substances, and now it is shown that the root part of it has the potential, too [30]. Tere were some critical compounds like cafeine [31], palmitic acid [32], oleic acid [33], and linoleic acid [34] in the EELR, most of which have proven regenerative potentials. As an example, oleic acid was shown to have anticancer and antiproliferative characteristics [23]. Moreover, palmitic acid was shown to have antiproliferative potentials in an earlier study [35]. Also, after 9 weeks, no adverse efects from the administration of the EELR were seen in any dog.
Regarding the hematological parameters, it was observed that the EELR in both doses didn't cause a signifcant change between the dogs with BPH and the healthy dogs, as it had in the previous studies [1,36]. Tis issue demonstrated that there was no blood toxicity following the consumption of the   Evidence-Based Complementary and Alternative Medicine 5 EELR. In the matter of the serum indices, after the platelet level in the BPH group was increased compared to the control group, a decrease in the EELR group was observed that could be the result of the natural regenerative compounds of the EELR (Table 1). Also, the PCT, MPV, and PDW levels showed no signifcant diferences in the groups. Te same trend happened to the WBCs, and their percentages didn't change signifcantly (Table 4), which was similarly in line with the previous studies [1,36]. As was seen in an earlier study [1], the level of albumin, as the main protein of plasma and one of the general health indices of the body, wasn't signifcantly correlated to the BPH, and despite a slight decrease in the BPH group compared to the control group, it didn't change signifcantly in this study (Table 5). Te number of liver enzymes in the BPH group was increased compared to the control group, which indicated that BPH can afect those indices. However, the changes were not signifcant; thus, they did not have a huge impact. Even that slight elevation of the liver indices could be caused by the natural cell-damaging efects of BPH [36]. In addition, the anti-infammatory potential of insulin was observed [37]. However, despite the nature of BPH, which is a kind of asymptomatic infammatory prostatitis [38], no sign of a signifcant alteration of insulin was observed in this research. A possible reason for that could be related to the substances in EELR, which include a lot of sugars and eventually downregulate the insulin to keep the serum glucose level steady. Fortunately, despite the moderate changes in insulin levels, no signifcant change was observed in glucose levels across all groups.
As the previous study said [1], the efect of BPH on renal parameters was undeniable, as was observed in this study. However, the changes were not signifcant between the control, the BPH, and the EELR groups. It was expected that the BUN level would increase signifcantly in the BPH group compared to the control group [36], but nothing was seen (Table 6). Also, the other possible reason for that issue could be the dogs' lack of a nutritious diet. After all, it is obvious that the BPH can elevate the kidney indices, just not enough to be concerned about them. It could hardly be seen that the creatinine and uric acid levels were increased in the BPH group compared to the control group, which meant that some renal damage occurred, but again, not too much to be concerned about [39]. It is also worth mentioning that the amounts of calcium and phosphorus were not changed signifcantly.
Te levels of both LDH and CPK markers in the BPH group were increased compared to the control group, which indicated that the BPH could cause cell destruction and muscle damage [40]. However, consumption of the EELR caused an increase in both of these markers, which could be the result of cell regeneration or reduced cell destruction.
Te most important indicator for detecting and staging BPH is the PSA level, which was signifcantly higher in the BPH group compared to the control dogs (P < 0.0001) as was shown in the previous study [25,41]. In the EELR20 treatment group, we saw a signifcant decrease (P < 0.01) in the PSA level compared to the BPH group. It seems that the EELR, with its antioxidant [42] and regenerative [43] properties, was able to reduce the amount of PSA in sick dogs by afecting the prostate tissue [44].
Te levels of both estradiol and testosterone were signifcantly decreased in the BPH group compared to the control group. Both of these hormones play a key role in BPH-patients [45]. Te estradiol was decreased because of the probable rise of the inhibitory pathways. As it was seen, the testosterone level was fallen due to the BPH, which caused the stimulating cells to proliferate and stop    Evidence-Based Complementary and Alternative Medicine functioning correctly, and at the same time trigger the signaling pathways that led to the inhibition of the estradiol [46,47]. Reduction of the testosterone level can also cause spermatogenesis dysfunction, which eventually leads to a worse clinical prognosis [48]. It should be noted that both of these hormones are important sex hormones, which were signifcantly elevated in the EELR20 group (P < 0.0001) due to the regenerative potentials and antioxidant properties of the EELR. Finally, the serum MDA level was examined to estimate the amount of oxidative abnormality, which was signifcantly higher in the BPH group compared to the control group due to the cell destruction and tissue stress caused by reducing the ROS, which was consistent with the previous study [49]. However, that increase was downregulated by the EELR20 administration signifcantly due to the natural antioxidants in the EELR. In addition, the TAC level had a negative correlation with the MDA level, and it was signifcantly lower in the BPH group compared to the control group (P < 0.05) [50,51]. Similarly, a signifcant rise in the TAC level was observed in the EELR20-treated dogs, which demonstrated a shining antioxidant potential. Based on earlier research, it was observed that tadalafl, as a strong antiproliferative agent, was not able to regulate the infammatory mediators in the BPH dogs [52], while the EELR, as a natural agent, managed to positively reduce ROS levels. It has been shown before that natural substances can effectively reduce oxidative stress and infammation [22].

Conclusion
Overall, it can be stated that the EELR can reduce the rate of prostate lesions in BPH efectively because of its antiinfammatory and antioxidant properties, which are boosted by its natural favonoid compounds, and also because it a signifcant level of omega-6 fatty acids. Regulating renal function in the excretion of substances and serum markers was another result of the administration of EELR. Finally, no toxicity or adverse efect was observed in the dogs at either dose.

Data Availability
Te data are available on a reasonable request from the corresponding author.

Conflicts of Interest
Te authors declare that there are no confict of interest.