Safety Assessment and Hepatic–Renal Protection of Cajanus cajan (L.) Millsp. Root and Its Soy Isoflavone Contents

Cajanus cajan (L.) Millsp., also known as pigeon pea, has roots that have exhibited much pharmacological potential. The present study was conducted to assess the safe dose of the ethanolic extract of C. cajan roots (EECR95) and to analyze the main soy isoflavones contents. In vitro, we investigated the mutagenicity and cytotoxic effect of EECR95 on Salmonella typhimurium-TA98 and TA100 (by Ames tests) and RAW 264.7, L-929, and HGF-1 cell lines (by MTT tests) for 24 h of incubation. We found no mutagenic or cytotoxic effects of EECR95. After administration of 0.2 or 1.0 g/kg bw of EECR95 to both male and female Wistar rats for 90 days, there were no significant adverse effects on the behaviors (body weight, water intake, and food intake), organ/tissue weights, or immunohistochemical staining, and the urine and hematological examinations of the rats were within normal ranges. EECR95 potentially decreases renal function markers in serum (serum uric acid, BUN, CRE, and GLU) or liver function markers (cholesterol, triglyceride, and glutamic-pyruvate-transaminase (GPT)). We also found that EECR95 contained five soy isoflavones (genistein, biochanin A, daidzein, genistin, and cajanol), which may be related to its hepatorenal protection. Based on the high dose (1.0 g/kg bw) of EECR95, a safe daily intake of EECR95 for human adults is estimated to be 972 mg/60 kg person/day.


Introduction
Traditional medicines, also known as herbal medicines, are raw, fresh, and dried extracts and whole dried plants, including roots, seeds, leaves, fruits, flowers, etc.; they have contributed to the development of the economy, health care, and pharmaceuticals [1].Herbal medicines have been reported to have various beneficial effects, and they are economical, cheap, available, safe, and low in side effects and toxicity; however, safety assessments are necessary to confirm the safe dose for the consumption of herbal products [2].The toxicity of herbs is related to the chemical constituents present in these plants; this toxicity may also contribute to acute or chronic, mutagenic, or carcinogenic effects [3].Toxic plants may affect different or multiple organ systems.For example, daily doses of aqueous extracts of Aphania senegalensis leaves (1000 to 2000 mg/kg) used to treat humans may cause liver toxicity; Herniaria cinerea is toxic and may cause digestive and alveolar Toxicity and genotoxicity of EECR95 were performed using the Ames test, as described by Maron and Ames (1983) and Vijay (2018) [20,21], with some modifications.Both toxicity and genotoxicity assays were performed with S. typhimurium strains TA98 and TA100.EECR95 (0, 0.25, 0.5, and 1.0 mg/plate) were used for both toxicity and genotoxicity assays.For the toxicity assays, we briefly incubated 0.1 mL of EECR95 + 0.1 mL of PBS + 0.1 mL of nutrient broth TA98 and TA100 strains at 37 • C overnight.The prepared agar contained 0.05 mM of L-Histidine, 0.05 mM of Biotin, and 0.09 M of NaCl.The plates were coincubated with the bacterial strains and prepared concentrations of EECR95, and agar was incubated at 37 • C for 48 h in the dark.The revertant colonies were counted.
Genotoxicity assays were determined similarly using the same bacterial strains and the same EECR95 concentrations with and without S9 fraction (9000× g supernatant in liver homogenate).4-nitroquinoline-N-oxide (4-NQNO, Sigma, St. Louis, MO, USA) was used as a positive mutagen without S9, and 2-aminoanthracene (2-AF, Sigma, St. Louis, MO, USA) was used as a positive mutagen with the S9 experiment.After incubation of the inverted plates at 37 • C for 48 h in the dark, the revertant colonies were counted.If the number of His + revertants/plate in the test group is more than twice that of the control group, it indicates mutagenicity or genotoxicity.

Animal Experimental Designs
All experimental animal protocols in this study were referred to Song et al. (2016) [22] and conducted in accordance with the Council of Agriculture, Executive Yuan guidelines.This experiment was approved by the Institutional Animal Care and Use Committee (IACUC, no.108017) of the Da-Yeh University.Male and female Wistar rats (6-8 weeks old, 200-225 g) were obtained from the National Laboratory Animal Center (NLAC) of Taiwan.Rats were fed (LabDiet 5001 Rodent Diet; PMI Nutrition International) and water was provided ad libitum under the normal conditions of a 12:12 h light: dark cycle at 22 ± 3 • C throughout this study (a total of 90 days).Rats were acclimatized for 1 week before experimentation and divided into the following three experimental groups using randomization.Female or male rats were randomly divided into 3 groups (6 animals in each group): group 1: control (CON); group 2: low-dose 95% ethanol extract of C. cajan (L.) Millsp.roots (p.o.0.2 g/kg bw, L-EECR95); and group 3: high-dose 95% ethanol extract of C. cajan (L.) Millsp.roots (p.o.1.0 g/kg bw, H-EECR95).EECR95 was prepared in saline.Rats were given daily gastric feeding (10 mL/kg bw) of EECR for 90 consecutive days.The body weight, food intake, and water intake were measured daily through 90 days.At the end of the experiment, all rats were sacrificed by carbon dioxide.Rats were euthanized by CO2 according to the AVMA Guidelines for the Euthanasia of Animal, and the CO2 flow rate was gradually increased from 30 to 70% of the cartridge volume/minute.The blood and organs of rats were collected for further examination.

Histopathological Studies
Ten animal organs, including the liver, kidney, lung, heart, spleen, brain, thymus, testes, ovaries, and adrenal glands, were collected and weighed at the end of the experiment.For the histopathological examinations, tissues were fixed in 10% buffered formalin and dehydrated in a graded series of alcohol, cleared in xylene, and embedded in paraffin wax.Multiple sections from each block were prepared at 5 µm and stained with haematoxylin and eosin kits (ab245880, Abcam, Cambridge, CB2 0AX, UK).

Detection of Soy Isoflavones Compound
The composition of soy isoflavones was carried out using an Agilent 1200 reversed phase High-performance liquid chromatography coupled with a diode-array detector (Hitachi, Chiyoda City, Japan, Chromaster 5430).A HIQ Sil C18W reversed-phase column was used (4.6 mm × 250 mm, 5 µm).The results were expressed in mg/100 g.Soy isoflavones (daidzin, daidzein, genistin, genistein, and cajanol) were measured according to the method presented in Vo et al. [11].

Statistical Analysis
All statistical analyses were performed using SPSS for Windows, version 17 (SPSS, Inc., Chicago, IL, USA).Data are expressed as means ± SD and analyzed using one-way ANOVA followed by Duncan's multiple range tests.p < 0.05 is considered statistically significant.

Effect of EECR95 on Bacterial Toxicity
The test doses caused cytotoxicity for the Ames strains TA98 and TA100, as evaluated by observing the colony forming units.According to Table 1, treatments of low-or high-dose EECR95 were not toxic on TA98 or TA100 strains, and the CFU values are not significantly different in all groups.Thus, low-and high-dose EECR95 had no bacterial toxicity effects.

Effects of EECR95 on Genotoxicity Tests
All groups' sample were tested for the mutagenic potential of S. typhimurium TA98 and TA100 in the absence and presence of S9 mix activation.Table 2 shows that none of the doses tested were mutagenic compared to the positive control (direct or indirect mutagenic test).

Cytotoxicity Effects of EECR95
We evaluated the cytotoxicity of three different cells (RAW 264.7,L-929 and HFG-1 ) treated with different concentrations of EECR95 (10-1000 µg/mL) using MTT assay (Table 3).EECR95 at concentrations up to 1000 µg/mL did not significantly affect to the cell viability of RAW 264.7,L-929, and HFG-1 cells (p < 0.05).Cells were pretreated with EECR95 (10-1000 µg/mL) and then incubated for 24 h.The viability was measured by MTT assay.The results are expressed as means ± SD; values (n = 3) in each column not sharing the same superscript letter are significantly different (p < 0.05).

Measurements of Body Weight and Dietary and Water Intakes
Male and female rats' behaviors were observed twice daily.As shown in Table 4, the body weight and food and water intake of male and female rats were not significantly different throughout the 90-day experiment (p < 0.05).  2 The results are expressed as means ± SD; values (n = 3) in each column not sharing the same superscript letter are significantly different (p < 0.05).

Urine Biochemical Tests
The urinalysis included total urine, color, specific gravity, clarity, protein, urobilinogen, pH, ketone, bilirubin, glucose, nitrite, occult blood, RBC, WBC, and epithelial cells.These abnormal urine ratios were not significantly different between the dose groups of female and male rats compared with the control group (Table 5, p > 0.05).

Hematological Tests
To investigate the effect of low or high EECR95 on the blood compositions of male or female rats, this was measured using an automatic serum biochemical analyzer.As shown in Table 6, the parameters (RBC, WBC, Hb, HCT, MCV, PLT, MCH, and MCHC) were not significantly different compared with those of male or female rats fed the blank diets (p > 0.05). 1 Red blood cells (RBC), white blood cells (WBC), hemoglobin (Hb), hematocrits (HCT), erythrocyte mean corpuscular volumes (MCV), platelet counts (PLT), mean corpuscular hemoglobin (MCH), and mean corpuscular hemoglobin concentration (MCHC) were determined using an automatic serum biochemical analyzer.L-EECR95 (0.2 g/kg bw); H-EECR95 (1.0 g/kg bw). 2 The statistical analysis exhibited no significant differences among groups. 3fL= 10 −15 L; 4 pg = 10 −12 g. a medical terminology.

Markers of Renal Function Tests
As shown in Table 7, the serum of rats fed with EECR95 for 90 days was collected and measured for renal function markers, including UA, BUN, CRE, GLU, and ALB.The results indicated that UA and GLU markers (male and female) of the control groups were significantly increased compared with the normal range.However, there was a trend towards decreased levels of all these markers in H-EECR95-fed male and female rats, but only serum markers in H-EECR95-fed females had statistically significant differences, whereas only UA in the serum of H-EECR95-fed males had significant differences compared to the controls (p < 0.05).

Markers of Liver Function Tests
Clinically, liver function tests in rats included CHOL, TG, TP, GOT, and GPT.Table 8 shows that both male and female rats in the control group had higher levels of CHOL, TG, and GPT levels above the normal range.However, CHOL, TG, and GPT levels were significantly lower in both male and female rats fed H-EECR95 compared to the control group (p < 0.05).The rat organs (heart, liver, spleen, lung, kidney, adrenal glands, brain, and testicles/ovaries) were collected and weighed; however, neither male nor female rats were significantly different after feeding low and high levels of EECR95 for 90 days (Table 9, p > 0.05).

Histopathology Records
Histopathologic studies showed no significant pathological changes in the adrenal glands, thymus, or lungs of the groups of rats fed L-EECR95 or H-EECR95 or of the control rats (Table 10).Histopathologic sections of the brain showed pathological changes, such as postmortem changes in female rats, ranging from multifocal, mild to severe/high; however, the effects were attenuated in female rats fed L-EECR95 and H-EECR95 compared with controls.In addition, histopathological sections of the kidney and liver also showed pathological changes, including chronic progressive nephropathy and necrosis that were multifocal and minimal to moderate/severe; however, there were no significant differences among the male rats' groups.As each group of rats had some occasional and minor lesions (heart, spleen, and testis/ovary), no specific changes can be attributed to the effect of the test substances.-No effect. 1 Pathological incidences: affected rats/total examined rats (n = 8). 2 The degree of lesions was graded from 1 to 5 depending on the severity: 1 = minimal (<1%); 2 = slight (1-25%); 3 = moderate (26-50%); 4 = moderate/severe (51-75%); 5 = severe/high (76-100%).L-EECR95 (0.2 g/kg bw); H-EECR95 (1.0 g/kg bw).

Discussion
Previous studies have reported that genistein and daidzein show no mutagenicity in the bacterial gene mutation test on S. typhimurium TA98 and TA100 strains (Ames tests) [24][25][26].The genotoxicity assay results indicated that EECR95 at a very high concentration (1.0 mg/plate) did not increase the number of histidine revertant colonies over the negative control in the TA100 and TA98 tester strains, either with or without S9 metabolic activation.The standard mutagens used in this study (4-NQNO and 2-AF) induced a clear positive response.The above results indicate that EECR95 was not mutagenic in this assay.The absence of mutagenicity for EECR95 in the tested S. typhimurium strains indicates that EECR95 does not affect the structural integrity of DNA.In addition, we also investigated the cytotoxic effects evaluated by MTT assay of EECR95 at concentrations from 10 to 1000 µg/mL.As the results showed, the highest doses of EECR95 (1000 µg/mL) had noncytotoxic effects on RAW264.7,L-929, and HGF-1 cells; their percentage of viable cells was more than 90%.Therefore, in vitro bacterial mutagenicity and cytotoxicity assays have confirmed that EECR95 is not mutagenic or cytotoxic at high doses.
Many recent studies have shown that changes in body weight are a simple and sensitive predictor of the effects of extracts; abnormal increases or decreases in body weight can indicate the degree of toxicity of drugs and chemicals [27,28].We found no statistical differences in the body weight or food and water intake of female and male rats fed low or high doses of EECR95 compared to the control group (p > 0.05).Therefore, we obtained preliminary evidence for the use of the highest safe dose for consumption (1.0 g/kg bw) of EECR95.
Clinicopathologic (urine biochemical and hematological analyses) results showed no statistically significant differences (p > 0.05) between female and male rats fed EECR95 compared to the controls.As with other organs and systems in the human body, urine biochemical analysis is the most basic test for the routine examination of urinary system function.The results of the urine analysis showed that no RBC, WBC, glucose, proteins, or hematuria were detected in the urine of female or male rats fed low or high doses of EECR95.The presence of hematuria is associated with infection, inflammation, trauma, hemorrhage, urolithiasis, toxemia, etc.Therefore, low and high doses of EECR95 did not cause infection or inflammation in either the female or male rats.Hematological analysis is considered an important element in toxicity studies and has been elucidated as a pathological reflection of pharmacological reactions, pathogenic processes, or normal biological processes [29].Consumption of toxic plants or agents can cause alterations in hematological characteristics [30,31].Delclos et al. conducted a study in which rats were impregnated with soy and alfalfa-free diets at doses of 0, 5, 25, 100, 250, 625, or 1250 ppm (genistein and daidzein) and showed that in any clinical chemistry or hematological parameter, there were no significant treatment-related differences in measurements [32].Yangzom et al. also reported no significant differences in hematological parameters between two isoflavones (e.g., kaempferol and biochanin A) administered orally to mice for 28 days [33].The results of this study showed that there were no statistically significant differences in various hematological parameters (including red blood cells (RBC), white blood cells (WBC), hemoglobin (Hb), hematocrit (HCT), mean red blood cell volume (MCV), platelet count (PLT), mean hemoglobin (MCH)m and mean hemoglobin concentration (MCHC)) between female and male rats administered with either low doses or high doses of EECR95, as compared to the control group (p > 0.05).
The kidneys play an important role in the excretion of wastes and toxins, such as urea, creatinine, and uric acid; the regulation of extracellular fluid volume, serum osmolality, and electrolyte concentrations; and the production of hormones, such as erythropoietin, 1, 25 dihydroxyvitamin D, and renin.Markers of renal function help to diagnose clinical disease and determine the progression of renal disease.Uric acid, BUN, CRE, and ALB are commonly used to measure renal function.As shown in Table 7, the serum BUN, CRE, and ALB values of the control rats were mostly within the normal range, but the serum uric acid and GLU values of the control rats were significantly higher than the normal range.However, the serum uric acid and GLU values of both the male and female control rats were significantly decreased after feeding L-and H-EECR95.
According to the National Institutes of Health, the overall prevalence of chronic kidney disease (CKD) is about 14%, and the most common causes of CKD are high blood pressure and diabetes [34].Uric acid has a role in the development of abnormal glucose metabolism by causing insulin resistance, impaired insulin secretion, and beta-cell dysfunction.Thus, hyperuricemia conditions are implicated in the pathogenesis of diabetes [35].In the past, we demonstrated the hypoglycemic potential of EECR95 by inhibiting the activities of key carbohydrate digestive enzymes (α-amylase and α-glucosidase) and anti-glycation (AGEs formation) [12].In addition, Yang et al. (2022) also found that EECR95 had a protective effect against methylglyoxal (MGO)-induced insulin resistance (IR) and hyperlipidemia in male Wistar rats [14].The present study also supports these findings and confirms the ability of EECR95 to protect the kidneys and prevent diabetes by lowering uric acid and GLU in the renal serum of male and female rats.
In the present study, serum liver function indices (e.g., CHOL, TG, and GPT) were not within the normal range in the control rats.However, the levels of CHOL, TG, and GPT were significantly decreased (p < 0.05) (that is, the liver function markers were significantly improved) after the administration of low-dose or high-dose EECR95 (Table 8).In the past, there have been considerable studies confirming the hepatoprotective effects of flavonoids.Soy isoflavones (e.g., genistein, soy isoflavones, bioflavonoid A, and formononetin) have been shown to have a protective effect against liver and kidney injury [36][37][38][39].Elmarakby et al. remarked that genistein (10 mg/kg, i.p. three times a week for 10 weeks) exerted renal-protective properties related to reduced renal inflammation, oxidative stress, and apoptosis in diabetic mice [36].Daidzein also possesses effects on oxidative stress and inflammation and the mediation of the angiotensin AT1 and Mas receptors in a fibrotic model of kidney disease of ovariectomized (OVX) rats, suggesting that daidzein can be able to replace estrogen for therapy in postmenopausal or older women against postmenopausal kidney damage [37].In addition, biochanin A (10 mg/kg and 20 mg/kg) was found to be protective against acetaminophen-induced hepatotoxicity in mice by inhibiting oxidative stress pathways and attenuating hepatic inflammation [38].
Bara ńska et al. indicated that the influence of soy isoflavones on CHOL and GLU levels as well as the modulation of lipid profiles, suggests benefits in preventing cardiovascular disease and type 2 diabetes [40].Soy isoflavones have been found in the stems and roots of pigeon pea, consisting of biochanin A, formononetin, genistein, cajanol, 2hydroxygenistein, and cajanin [19].However, the presence of genistein, daidzein, and cajanol has already been reported previously for C. cajan roots [11,13]; genistin is a glycoside form of genistein and is mainly found in soy-derived foods.Kwon et al. indicated that the oral bioavailability of genistin is greater than that of genistein [41].Therefore, we further analyzed the content of biochanin A, cajanol, genistein, daidzein, and its glycosides (genistin and daidzein) in EECR95 using an HPLC-DAD-UV/Vis system.
The results of the HPLC chromatogram indicated that five soy isoflavones (daidzein, genistin, genistein, cajanol, and biochanin A) were found in EECR95.Thus, this study demonstrated that C. cajan (L.) Millsp.roots contain soy isoflavones, which exhibit several pharmacological properties.Therefore, EECR95 was effective in lowering lipids, cholesterol, and GPT, suggesting its hepatoprotective effects, and its main potent components were hypothesized to be related to flavonoids.
The immunohistochemical (IHC) staining results provided conclusive evidence of organ toxicity and correlated with changes in biochemical tests.The analysis of pathological examinations by IHC staining showed no histopathological changes in organs or tissues (brain, heart, liver, spleen, lungs, kidneys, thymus, and adrenal glands (ovaries, testes)) of male and female rats fed EECR95 continuously for 90 days at low or high doses (0.2 or 1.0 g/kg/day).Therefore, based on calculations from the literature [42], we estimated a no-observed adverse effect level (NOAEL) value for EECR95 of approximately 1.0 g/kg bw extrapolated from rats to humans, which corresponds to approximately 972 mg/60 kg man/day.

Conclusions
To the best of our knowledge, our study is the first study to examine the safety assessment of C. cajan.EECR95 did not exhibit any toxic effects in mutagenicity (0.25-1.0 mg/plate) or cytotoxicity (10-1000 µg/mL) in in vitro assays or in in vivo safety assays in rats fed continuously for 90 days (0.2-1.0 g/kg body weight), suggesting that EECR95 should be reasonably safe for human application.Based on the NOAEL for EECR95, we calculated that the safe human dose should be 972 mg/60 kg person/day.In addition, we found that EECR95 was able to reduce liver and kidney function markers, especially in lowering uric acid, lipid, cholesterol, and blood glucose levels, suggesting it has great potential for the development of nutraceuticals.Furthermore, this study showed that EECR95 contains five soy isoflavones known to have estrogenic effects (genistein, biochanin A, daidzein, genistin, and cajanol), which is hypothesized to be a major component of EECR95's ability to protect the liver and kidneys.

Table 4 .
Body weights and dietary and water intakes of male and female rats fed low and high levels of EECR95 for 90 days.

Table 5 .
Number of abnormal urine tests of male and female rats fed with EECR95 for 90 days.

Table 6 .
Blood compositions of male and female rats fed with EECR95 for 90 days.

Table 7 .
Renal function markers in serum of male and female rats fed with EECR95 for 90 days.

Table 8 .
Liver function markers in serum of male and female rats fed with EECR95 for 90 days.

Table 9 .
Relative organ weights in male and female rats fed low and high levels of EECR95 for 90 days.

Table 10 .
Summary of pathological incidence 1 of EECR95 in 90 days' feeding toxicity in male rats.