Abstract

This study was aimed at assessing the safety of a mixed extract of fenugreek seeds and Lespedeza cuneata (YHM), which effectively relieves male menopausal symptoms. To this end, male and female Sprague-Dawley rats were divided into the following groups and repeatedly administered YHM orally for 90 days: control, low-dose (500 mg/kg/day), intermediate-dose (1,000 mg/kg/day), and high-dose (2,000 mg/kg/day) groups. The animals were monitored for general symptoms; their body weights and electrolyte levels were measured; and urinalysis, blood chemistry and biochemistry tests, and histopathological tests were performed to assess the toxicity of YHM. The no-observed-adverse-effect level of YHM was 2,000 mg/kg/day for all male and female rats. While in the YHM-administered and control groups, most parameters were within the normal range; some rats in the high-dose group showed changes not induced by the test substance but which may be specific to an individual animal or may occur naturally. Thus, on the basis of our findings, we consider that YHM may be a safe, non-toxic substance for alleviating male menopausal symptoms.

1. Introduction

In 2017, the elderly, i.e., individuals aged ≥ 65 years, accounted for 13.8% of South Korea’s total population, and this percentage is expected to reach 20.3% in 2025. ¹ The incidence of hormone-related disorders such as menopausal symptoms, sexual dysfunction, and prostatic hypertrophy is expected to increase with the transition from an aging to an aged society. Female menopausal symptoms are easy to identify and manage owing to markedly reduced levels of estrogens, which are female sex hormones, which occur with aging. Male menopausal symptoms, on the other hand, are harder to identify and are often managed late since the levels of testosterone, which are male sex hormone, decrease gradually. In general, testosterone levels in men start to decrease by 3.1-3.5 ng/dL per year from the age of 30 years. ^{2, 3} Male menopause refers to the period after the age of 30 years during which various symptoms develop because of an aging-induced reduction in blood testosterone levels. These symptoms include low energy, erectile dysfunction, reduced muscle mass, abdominal obesity, and anxiety. ^{4, 5}

Fenugreek is an annual herb in the legume family; it originated from the Mediterranean region and North Africa, and its seeds have traditionally been used for medicinal purposes. ⁶ Fenugreek seeds have been used to treat kidney and bladder symptoms for a long period and have been recently found to increase energy, maintain blood glucose and insulin balance, and aid weight management. ^{7, 8} Fenugreek seed extract has been reported to positively affect sexual health and quality of life through its anabolic activities and androgen activation. ⁹ The beneficial effects of the extract are attributed to the extract’s ability to increase testosterone levels. Fenugreek seed extracts may effectively treat menopausal symptoms in older men and have been reported to improve male menopausal symptoms by increasing the total and free testosterone levels by inhibiting α-reductase and aromatase. ¹⁰ Lespedeza cuneata, also known as Chinese bushclover, is a plant of the legume family. It has been used in herbal medicine to treat lack of energy, premature ejaculation, coughing, and asthma. ¹¹ In particular, L. cuneata can exert endothelial cell-dependent vasorelaxing effects through the nitric oxide-cGMP system, and the n-hexane layer of methanol extract of L. cuneata elicits a relaxation effect on the corpus cavernosum smooth muscle of rabbits. ^{12, 13} Therefore, L. cuneata has been used to treat cardiovascular disease and erectile dysfunction.

While numerous studies have proven the beneficial health effects of newly developed or existing substances, they have been mostly limited to verifying only the positive effects of the active ingredients. Few studies have assessed the toxic effects of active ingredients when ingested. Hence, in this study, we assessed the safety of YHM, which is a mixed extract of fenugreek seeds and L. cuneata that has been found to relieve male menopausal symptoms, by conducting a toxicity test through oral administration to rats, following the Good Laboratory Practice (GLP). ¹⁴

2. Materials and Methods

YHM, in a powdered form, was provided by DuhanBio Co., Ltd. (Seoul, Korea) for the experiment. The extract was prepared by performing heat extraction of dried fenugreek seeds and L. cuneata stems and leaves by using 70% ethanol, filtering the extracts using a filter press, and concentrating them. Each extract was prepared in powdered form by using a pilot spray dryer and mixed to obtain YHM. YHM was diluted in water for oral administration prior to use in the experiment.

Five-week-old male and female Sprague-Dawley rats were purchased from DBL Co., Ltd. (Eumseong, Korea). The experiment was conducted at the SPF Laboratory Animal Facility of the GLP Center at Daegu Catholic University (16-REO-003). Two rats were kept per polycarbonate breeding cage (280 mm [W] × 420 mm [L] × 200 mm [H]) at a temperature of 22 ± 3°C, relative humidity of 22 ± 3 °C, and light intensity of 150-300 Lux in a 12/12 h light/dark cycle. During one week of acclimatization, the animals were monitored for any abnormal symptoms. Only animals deemed to be normal were used in the experiment. Sterilized, solid laboratory animal feed and sterilized tap water were provided ad libitum.

The laboratory animals were divided into control and YHM-administered groups, with 10 animals per group. Each group was further divided into male and female groups. Ten rats were randomly allocated to a group such that their body weights were close to the mean body weight of the group (Table 1). The animals were identified by tail marking. ¹⁵ Considering that the test substance was a natural extract, the administration dose was set to 2,000 mg/kg/day for the high-dose group, which is greater than the standard dose limit for repeated-dose toxicity tests (1,000 mg/kg/day); 1,000 mg/kg/day for the intermediate-dose group; and 500 mg/kg/day for the low-dose group. Thus, the animals were divided into four groups, including the control group.

The test substance was prepared at concentrations of 2,000, 1,000, and 500 mg/10 mL/kg using water as the solvent for oral administration. Using a syringe with a zonde for enteral administration, the test substance was directly administered into the stomach. The volume administered was calculated as 10 mL/kg, based on the most recently measured body weight of the animals. The test substance was administered once daily for 90 days.

All animals were monitored once daily for the type, onset, and severity of general symptoms and twice a day for fatal conditions or death. The animals were monitored for the entire course of YHM administration (90 days). Body weights of all animals were measured on the first day of YHM administration, once a week thereafter, and once on the day before the autopsy and on the day of the autopsy.

Urinalysis was conducted for all five animals in each group 13 weeks after YHM administration. Fresh urine (1 mL) collected from rats in metabolic cages for 3-4 h was used for the general analysis and urine sediment analysis. The total urine volume was measured based on the amount of urine that was continuously collected for 24 h.

The rats were fasted for over 17 h before the day of the autopsy and anesthetized via isoflurane inhalation. Blood samples were collected from abdominal arteries. An autopsy was conducted on all organs, and the findings were recorded.

A portion of the blood collected during the autopsy was added to a vacutainer tube (Vacutainer, BD, USA) containing EDTA-2K, an anticoagulant. General blood test parameters (White blood cell count [WBC], red blood cell count [RBC], hemoglobin concentration [HGB], hematocrit [HCT], mean corpuscular volume [MCV], mean corpuscular hemoglobin [MCH], mean corpuscular hemoglobin concentration [MCHC], red cell distribution width [RDW], hemoglobin distribution width [HDW], platelet [PLT], mean platelet volume [MPV], and reticulocyte [RET]) and leukocyte (neutrophil [NEU], lymphocyte [LYM], monocyte [MONO], eosinophil [EOS], basophil [BASO], large unstained cells [LUC]) counts were measured using an automated hematology analyzer, K-96 (ADVIA 2120, Siemens, USA).

For serum biochemistry test, blood samples were added to vacutainer tubes containing clotting activators and allowed to coagulate at room temperature for 10–15 min. Next, serum was obtained by centrifuging the blood samples for 10 min at 4,000 rpm (MF80, Hanil, Korea) and then analyzed using an automated hematology analyzer K-97 (KONELAB 20XT, Thermo Fisher Scientific, USA). Electrolyte levels were measured using the K-99 electrolyte analyzer (744 Na+/K+/Cl- Analyzer, Siemens, USA).

For all animals, organs were harvested during the autopsy, and their weights were measured using a precision scale. The organs investigated in this measurement included adrenal, pituitary, testis, ovary, epididymis, thymus, prostate, spleen, kidney, heart, lung, brain, and liver.

The harvested organs were fixed in 10% neutral-buffered formalin. The fixed organs and tissues were subjected to a conventional processing procedure that involved trimming, dehydration, and paraffin embedding to prepare tissue sections. Sections were then cut and stained with hematoxylin and eosin. In a histopathological examination, all fixed organs and tissues from the control and high-dose groups were examined under a microscope. The organs and tissues studied in this examination included prostate, kidney, liver, spleen, lung, heart, and kidney. Organs observed as abnormal in these groups were also examined in the low- and intermediate-dose groups.

Mean values between the control and YHM-administered groups were compared using parametric or non-parametric multiple comparison methods. Statistical analyses were performed using the SPSS 19.0 (IBM, USA).

One-way analysis of variance (ANOVA) was used to evaluate the significance of the differences in the mean body weight, feed consumption, blood chemical and biochemical parameter values, and organ weights. If a significant difference was found, the homogeneity of variance was evaluated using Levene’s test. If the homogeneity of variance was satisfied, Duncan’s multiple range test was used. Dunnett’s t-test was used for heteroscedastic data.

Urinalysis results were expressed as severity after scale transformation as shown in Table 2, and statistical analysis was conducted. If significant differences were found in the Kruskal-Wallis H test, the Mann-Whitney U test was performed to confirm the statistical significance of the differences with respect to the control group.

3. Results

The criteria listed in Table 3 were used to determine toxicity. The no-observed-effect level (NOEL) is the maximum concentration at which a test substance does not cause toxic or pharmacological changes. The no-observed-adverse-effect level (NOAEL) is the maximum concentration at which a test substance does not induce adverse effects or lead to an evident disease. The lowest-observed-adverse-effect level (LOAEL) is the minimum toxic concentration at which a test substance induces adverse effects.

No deaths or unusual symptoms were observed in any group, including the control group, throughout the 90-day repeated oral dose toxicity study period. No significant differences in weight change were observed in any experimental group, compared to the control group (Table 4).

No significant findings and differences in urinalysis results were found between the experimental and control groups (Table 5). No unusual findings or significant differences in the general blood test results and reduction in leukocyte count were observed between the male experimental and control groups (Table 6). While the red blood cell counts were significantly reduced in the female high-dose group (2,000 mg/kg/day) compared to that in the control group, the reduction was small and within the normal range and thus deemed a change unrelated to the test substance (P < 0.05) (Table 6).

No unusual findings or significant differences in the results of the serum biochemistry test and electrolyte measurements were observed between the male experimental and control group. While the total cholesterol (CHO) level was significantly higher in the female high-dose group (2,000 mg/kg/day) than in the control group, the increase was small and within the normal range and thus deemed unrelated to the test substance (P < 0.05) (Table 7).

No unusual findings or differences in organ weights were observed between the male experimental groups and the control group. While the absolute weight of the liver significantly increased in the female high-dose group (2,000 mg/kg/day) compared to that in the control group, the increase was within the normal range and deemed unrelated to the test substance (P < 0.05). While the relative weight of the heart significantly increased in the high-dose group (2,000 mg/kg/day) compared to that in the control group, the increase was within the normal range and deemed unrelated to the test substance (P < 0.05) (Table 8).

Inflammatory cell infiltration, local inflammation, and congestion were observed in the prostate, kidney, liver, spleen, and heart of some animals in the male high-dose group (2,000 mg/kg/day). These symptoms were also observed in the control group and thus deemed unrelated to the test substance (Table 9). Inflammatory cell infiltration and local inflammation were observed in the kidney, liver, and heart of some animals in the female high-dose female group (2,000 mg/kg/day). The same symptoms were observed in the control group and thus deemed unrelated to the test substance (Table 9).

4. Discussion

YHM has previously been found to relieve various male menstrual symptoms by improving testosterone levels. ¹⁴ In this 90-day repeated oral dose toxicity study, the NOEL of YHM was determined to be 2,000 mg/kg/day for both male and female rats. The maximum recommended starting dose of YHM is 32.25 mg/kg/day or 1,935 mg/day for an adult weighing 60 kg. This is approximately five-fold higher than the proposed dose limit of 400 mg/day for an adult weighing 60 kg, which was approved by the Korean Ministry of Food and Drug Safety, and thus safe. ¹⁴

Male rats showed changes unrelated to the test substance, which determine the NOEL, in histopathological examinations. ¹⁶ Some rats in the high-dose groups showed inflammatory cell infiltration in the prostate and kidney; however, these symptoms were also observed in the control group. Furthermore, mild inflammation in the prostate and kidneys can occur naturally with aging. Since local liver inflammation, splenic congestion, and inflammatory cell infiltration in the heart observed in the experimental groups were also observed in the control group, these changes were not deemed to have been induced by the test substance.

Female rats showed changes unrelated to the test substance, which determine the NOEL, in the blood biochemistry test, organ weight measurement, and histopathological examination. ¹⁷ The serum biochemistry test revealed that the total CHO level increased by 26% in the high-dose group (2,000 mg/kg/day) compared to that in the control group but was still within the normal range. The absolute weight of the liver increased by 10.3% in the high-dose group compared to that in the control group but was still within the normal range. The histopathological examination revealed inflammatory cell infiltration in the kidney in one rat in the high-dose group. Inflammatory cell infiltration can also occur naturally with aging. A few rats in the high-dose group showed local liver inflammation; however, this was also observed in the control group. The overall test results for the experimental groups were within the normal range compared to those obtained for the control group; however, a few changes unrelated to the test substance were observed. These changes are not induced by the test substance, but are rather specific to certain animals or are natural phenomena. Based on these findings, we considered YHM to be a safe and non-toxic substance.

5. Conclusion

In this study, a repeated oral dose toxicity test was performed on rats to assess the safety of a mixed extract of fenugreek seeds and L. cuneata, YHM, which has been proven to relieve menopausal symptoms in men. No deaths or unusual symptoms occurred in any group, including the control group. No unusual findings were noted in terms of weight change, eye examination results, urinalysis results, feed consumption, blood test results, and histopathological examination results. All test results were within the normal range in the experimental group compared to those in the control group. In both the female and male high-dose groups administered YHM at 2,000 mg/kg/day, which is higher than the standard 1,000 mg/kg/day dose limit for repeated dose tests, the NOEL was determined to be 2,000 mg/kg/day, indicating that YHM is safe and non-toxic within a certain dose range. Thus, our findings suggest that YHM could be a safe, non-toxic substance for alleviating male menopausal symptoms.

Acknowledgements

This study was funded by DuhanBio Co. Ltd., Seoul, Korea.

References