|Year : 2023 | Volume
| Issue : 3 | Page : 293-308
Safety assessment of Libirite capsule (a polyherbal formulation) in experimental animals (Sprague Dawley rats and Swiss albino mice)
Vasant E Narke1, Sanjay U Nipanikar2, Ujwala V Khisti1, Sachin A Upasani2
1 Department of Toxicology, Bibwewadi, Pune, ToxIndia, India
2 Medical Services Department, Ari Healthcare Pvt. Ltd., Pune, Maharashtra, India
|Date of Submission||04-Aug-2022|
|Date of Acceptance||09-May-2023|
|Date of Web Publication||16-Aug-2023|
Dr. Sanjay U Nipanikar
Ari Healthcare Pvt. Ltd., Office at No., 107, 1st Floor, S.No.1, World Trade Center, Tower one, Opp. EON SEZ, Kharadi, Pune 411014, Maharashtra
Source of Support: None, Conflict of Interest: None
BACKGROUND: Libirite capsule (LC) is a polyherbal formulation, developed to treat erectile dysfunction and loss of libido. Present study aimed to perform the acute oral toxicity studies of LC in Swiss Albino mice and Sprague Dawley rats, and repeated dose subchronic 90-day toxicity study were performed to assess the safety of LC. METHODS: LC was orally administered at 2000 mg/kg to the animals as per Organization for Economic Cooperation and Development (OECD)-423 guidelines. In a repeated dose oral toxicity study, LC was administered through oral gavage in a dose of 250, 500, and 1000 mg/kg for 90 days and compared with control groups as per OECD-408 guidelines. Posttreatment changes in food consumption, body weight, and biochemical, hematological, and laboratory parameters were observed. RESULTS: LC did not produce any adverse or mortality events in animals during acute studies. In a 90-day toxicity study, rats exhibited no toxicity symptoms or death. No significant changes were found in hematological and biochemical parameters. No significant alteration was seen in organ and body weight. Microscopic findings were incidental and identical for control and treated animals at 1000 mg/kg. LC did not produce any histopathological changes in target organs. No change in the recovery group was observed when compared with the control group animals, which indicated a complete reversal. CONCLUSIONS: Median lethal dose50 of LC was observed to be more than 2000 mg/kg. No observed adverse effect level of LC was considered 1000 mg/kg.
Keywords: Acute, libido, Libirite capsule, erectile dysfunction, polyherbal, subchronic, toxicity
|How to cite this article:|
Narke VE, Nipanikar SU, Khisti UV, Upasani SA. Safety assessment of Libirite capsule (a polyherbal formulation) in experimental animals (Sprague Dawley rats and Swiss albino mice). J Drug Res Ayurvedic Sci 2023;8:293-308
|How to cite this URL:|
Narke VE, Nipanikar SU, Khisti UV, Upasani SA. Safety assessment of Libirite capsule (a polyherbal formulation) in experimental animals (Sprague Dawley rats and Swiss albino mice). J Drug Res Ayurvedic Sci [serial online] 2023 [cited 2023 Sep 23];8:293-308. Available from: http://www.jdrasccras.com/text.asp?2023/8/3/293/383694
| Introduction|| |
Erectile dysfunction (ED) or male impotence is an inappropriate penile erection sufficiently required for sexual activity. Various studies reported that around 15%–20% of men described some sexual problems., About 80% of cases of ED have an organic etiological factor behind them, which was once considered psychogenic.,, Androgen deficiency, atherosclerosis, hypertension, diabetes mellitus, hyperlipidemia, prostate issues, cardiac disorders, spinal cord injuries, penis anatomical deformity, social and psychological reasons, stress, and depression can cause male impotency.,
The management of male sexual impotence includes, but is not limited to, oral phosphodiesterase type 5 inhibitor drugs such as sildenafil citrate and intracorporeal agents with vasodilatory effects. However, prolonged usage of many of these drugs can cause major side effects, including cardiovascular complications.,, Many health agencies worldwide have recommended utilizing traditional plants to manage various disorders, because they are nontoxic with lesser side effects. Around the world, traditional herbal remedies are used to treat sexual disorders with potent aphrodisiac activity.,,,,,,,,,
Libirite capsule (LC) is a polyherbal formulation developed by Ari Healthcare Pvt. Ltd., Pune, India, for managing mild-to-moderate cases of ED and loss of libido. LC contains extracts of Tribulus terrestris L., Withania somnifera Dunal., Asparagus adscendens Roxb., Mucuna pruriens Baker.,Piper betle L., Myristica fragrans Houtt., Syzygium aromaticum (L.) Merr & M Perry, and powder of Crocus sativus L. Almost all the ingredients used in LC possess aphrodisiac (libido enhancer), antistress, and antidepressant properties.,,,,,,,,,,, These herbs help relax the penile tissue and, thus, maintain an erection. Also, these herbs help in alleviating psychological elements involved in sexual dysfunction.,,,, Though the utilization of traditional medicine has passed the test of time, it may produce various toxic adverse effects in new and higher dosage forms. Thus, there is a necessity for the evaluation of toxicity of such herbal formulations and raw materials.,,,,,,,,,,,,,,,,,,,,, Hence, this study evaluated the acute oral toxicity and repeated dose oral toxicity of LC in experimental animals.
| Material and Methods|| |
The method was designed, and studies were performed in a stepwise manner. Acute toxicity studies and subchronic 90-day toxicity studies were conducted after the approval of the Institutional Animal Ethics Committee (IAEC) meeting on January 23, 2016, with project number TI/101003. Experiments complied with the OECD (1998) principles of Good Laboratory Practice (GLP). LC’s acute oral toxicity studies were performed in compliance with the OECD-423, Section-4 guidelines; the repeated dose 90-day oral toxicity study of LC was performed as per OECD-408 guidelines.
Kits and Reagent
Analysis of biochemical parameters including alanine transaminase, aspartate transaminase, alkaline phosphate, serum creatinine, lactate dehydrogenase, serum sodium, serum potassium, and HDL was done using Biosystem kit. For the analysis of serum albumin, cholesterol, triglycerides, LDL, total protein and glucose Q-Line kits were used. Urine analysis was done using Uriscan kit.
Sprague Dawley (SD) rats of age from 9 to 11 weeks and Swiss Albino (SA) mice of age 7–9 weeks and female were selected for two separate acute toxicity studies (OECD-423 guidelines). SD rats of both sex of age from 7 to 9 weeks were selected for a 90-days oral toxicity study (OECD-408 guidelines). Animals were bred and tended at the animal house of ToxIndia, Pune, India. Female animals were nonpregnant and nulliparous. All animals were sustained and adapted under standard household conditions, viz. temperature was 22 ± 3°C, relative humidity was between 30% and 70% with 12–15 air changes per hour and 12-h light and 12-h dark cycle. Animals were selected randomly after the veterinary examination. All animals were fasted overnight before treatment. Food was offered 3 and 1 h after dosing in rats and mice, respectively. All the animals were provided clean water (passed through reverse osmosis water filtration and UV ray system) and Nutrimix brand pelleted standard rat and mice feed (M/s: Nutrivet Life Sciences, Pune, India) ad libitum.
All the experiments were conducted after approval with approval number TI/101003 dated January 23, 2016, of the IAEC meeting and as per the Committee for Control and Supervision on Experiments on Animals, India, for the care and ethical use of experimental animals and as per standard operating procedures currently in force at ToxIndia, Pune, India.
Preparation of herbal formulation
LC (test Item) was manufactured and provided by Ari Healthcare Pvt Ltd. The ingredients of LC were as follows: hydroalcoholic extracts of Gokshura (Tribulus terrestris L.) fruit 140 mg, Ashvagandha (Withania somnifera Dunal.) root 120 mg, Shveta Musali (Asparagus adscendens Roxb.) root 100 mg; aqueous extract of Kapikacchu (Mucuna pruriens Baker.) seed 90 mg; hydroalcoholic extracts of Nagavalli (Piper betle L.) leaf 40 mg, Jatiphala (Myristica fragrans Houtt.) fruit 20 mg, Lavanga (Syzygium aromaticum L. Merr. & M. Perry) flower bud 15 mg, and powder of Kesara (Crocus sativus L.) stigma 5 mg. Each hydroalcoholic extract contained approximately 40% and 60% of alcohol and water, respectively.
The test item (received from the sponsor) was in capsule form. The powder was removed from the capsule and used as a test item for dose preparation. The test drug concentration was prepared 200 mg/ml for the acute toxicity study. For 90 day oral toxicity study three drug concentrations (25, 50 and 100 mg/ml) were prepared for respective groups. Solutions were freshly prepared prior to dosing. The homogeneity was ensured using Magnetic Stirrer during dose administration. Animals were treated in sequence at 24-h intervals. However, the dosing intervals were determined by assessing the toxic sign’s duration, onset, and severity. According to historical data, the initial dosing level of 2000 mg/kg was selected from one of the four dosing levels, that is, 5, 50, 300, and 2000 mg/kg.
The dose levels for subchronic (90 days) oral toxicity were established upon the findings of the dose-ranging study. The dose of 1000 mg/kg of body weight was selected as the highest dose. Of these dose levels, the lowest dose was 250 mg/kg. The intermediate 500 mg/kg dose has been placed between the two dose levels.
The excess formulation was disposed of following the appropriate regulatory requirements and information supplied by the sponsor. Food was suspended overnight before the administration of the test item. Animals were allowed to access the water ad libitum.
Standard procedures were set for safe handling of the test drugs. Eye and skin shield equipment were used while dosing animals. The sponsor/manufacturer supplied information regarding the safety of the test item. Test drug substance was classified according to the Globally Harmonized System (GHS) for the classification of chemicals and predefined dosage method.
Acute oral toxicity study in SD rats and SA mice
Stepwise assessment of the toxicity of LC was done following the treatment of animals through oral gavage. Three female SD rats and three female SA mice were used per step. According to historical data of the test item, the initial dosing level of 2000 mg/kg was selected from one of the four dosing levels, that is, 5, 50, 300, and 2000 mg/kg. Food was suspended overnight before the administration of the test item. Animals were allowed to access the water ad libitum in both acute toxicity studies.
In step 1, animals were dosed with 2000 mg/kg of body weight. The treated animals were observed for clinical signs, toxicity, and mortality signs at 30 min, 1 h, 2 h, 3 h, 4 h, and 6 h, and after that once a day for 14 consecutive days. Additional parameters that display toxicity signals viz. changes in the appearance of the skin, mucus membrane, and eyes; changes in the behavior or activity of animals; or any systemic changes were observed (if necessary). Body weights of animals were recorded before dosing and on a weekly basis in acute toxicity studies. All animals were subjected to gross necropsy with internal and external observations of organs and cavities. No abnormal changes (if any) in targeted organs were noted during histopathological examination. After observation of clinical signs and mortality at step 1, the same dose was administered at step 2 on three more female animals (SD rats and SA mice) in both acute studies, respectively. All animals were observed for adverse signs and mortality for 14 days.
Ninety-days repeated dose oral toxicity study in SD rats
Based upon the dose-ranging study results, 10 female and 10 male SD rats were fed with LC through oral gavage on a daily basis with doses of 0 (control), 250, 500, and 1000 mg/kg of body weight till 90 days. Further, animals were euthanized and evaluated for toxicity findings. Simultaneously, control group animals were fed with distilled water at 10 mL/kg. Satellite group animals receiving distilled water at 10 mL/kg and LC at 1000 mg/kg levels were observed for 28 days and 90-day exposure to assess reversibility, diligence, or late toxicity incidences.
Initially, animals were examined for clinical signs. Toxicity and mortality signs were examined on a daily basis and after that during exposure and reversal periods. Weekly recording of body weight and food consumption was done. Hematological and biochemical investigations and urinalysis were done at the termination of the study. Animals were euthanized and evaluated for toxicity findings. Histopathological analysis was done on organ tissues of control and high-dose level group animals as mentioned in the trial protocol.
Changes in body weights, organ weights, food consumption and their ratios, biochemical and hematological parameters, and urine analysis parameters were assessed. Dunnet as a post hoc test and analysis of variance (one-way) test were used to analyze and compare the different treatment groups with the control group. Student t test was used to compare high-dose recovery and control recovery groups. All statistical parameters were assessed at a 95% confidence level (P < 0.05).
| Results|| |
Acute oral toxicity study
In step 1, LC at 2000 mg/kg dose did not induce any toxicity signs throughout the study. No mortality observations were found in SD rats and SA mice during the 14-day trial period in acute toxicity studies. During the 14-day posttreatment analysis period, the LC group was not shown any adverse gain in body weights of treated animals [Table 1] and [Table 2]. LC group was not shown any pathological alterations in the organs of animals during the necropsy [Table 3]. In step 2, similar results were observed in animals.
|Table 1: Body weight (g) summary of acute oral toxicity study in Sprague Dawley rats|
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|Table 2: Body weight (g) summary of acute oral toxicity study in Swiss albino mice|
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|Table 3: Individual animal fate and necropsy findings in acute oral toxicity study|
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Ninety-day repeated dose oral toxicity study
Clinical and mortality signs
There were no mortality incidences among the male and female rats in the LC group up to 1000 mg/kg dose levels. Rats fed with LC up to 1000 mg/kg dose showed no notable abnormal signs in either sex. Accidental diarrhea and urination were noted in a few animals of all groups, including the vehicle control group. It was clear that accidental diarrhea and urination were not because of drug consumption, as the weight and other vital signs of all the animals were within normal limits till the completion of the study.
Feeding and body weights
LC group has not shown any substantial and remarkable change in mean body weights of animals treated at and up to 1000 mg/kg dose levels [Table 4] and [Table 5]. In food consumption analysis, animals fed with LC at and up to 1000 mg/kg doses were compared with control group animals. Ocular findings were normal in control as well as high-dose groups.
|Table 4: Mean body weight (g) of male Sprague Dawley rats in 90-day oral toxicity study|
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|Table 5: Mean body weight (g) of female Sprague Dawley rats in 90-day oral toxicity study|
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Mean values of hematological analysis such as Hb%, total erythrocyte counts, packed cell volume, leucocyte counts, erythrocytes indices, platelet count, and clotting time in animals fed with LC at and up to the level of 1000 mg/kg, and reversal group (1000 mg/kg) did not shown any change at the end of the study (day 90) and at reversal period (day 118). Also, male and female rats treated with LC at and up to the dose of 1000 mg/kg did not exhibited any substantial change in hematological and biochemical parameters at the termination of the study [Table 6][Table 7][Table 8][Table 9]. Parameters evaluated during the urine analysis showed no remarkable treatment effect up to 1000 mg/kg. The reversal group exhibited normal values compared with the control and the parameters evaluated during the urine analysis.
After necropsy, mean values of absolute and relative organ weights viz. kidneys, adrenal glands, liver, spleen, brain, testes/ovaries, uterus, thymus, and heart of animals treated with LC at and up to 1000 mg/kg were comparable to respective control groups at treatment termination and reversal periods [Table 10] and [Table 11]. LC group animals did not show any substantial pathological alterations in any organ or tissues at and up to 1000 mg/kg and in the reversal group [Table 12].
Parameters evaluated during the urine analysis did not exhibit any remarkable treatment effect up to the 1000 mg/kg.
All microscopic changes noticed during histopathological analysis seemed incidental and identical for the control and the treated animals at and up to 1000 mg/kg. No histopathological changes were observed in rats fed with LC at and up to 1000 mg/kg doses under conditions as mentioned earlier [Table 13] and [Figure 1][Figure 2][Figure 3][Figure 4][Figure 5][Figure 6][Figure 7][Figure 8][Figure 9].
|Table 13: Summary of histopathological findings in 90-days oral toxicity study|
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|Figure 1: Histopathological representation of bronchial epithelium and alveoli|
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|Figure 2: Histopathological representation of centrilobular area and hepatic parenchyma|
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|Figure 3: Histopathological representation of glomeruli and renal tubules|
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|Figure 4: Histopathological representation of cardiac muscles and myocytes|
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|Figure 5: Histopathological representation of skeletal muscle fibers and myocytes|
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|Figure 6: Histopathological representation of seminal vesicle and mammary gland|
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|Figure 7: Histopathological representation of the uterus and prostate gland|
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No observed adverse effect level
As per the study results, the test item LC did not show any adverse event on overall body growth and animal health during the biochemical, hematological, neurological, and behavioral analysis of animals at and up to the dose level of 1000 mg/kg. It reveals that the LC did not show a significant toxicological change in any animal till 90 days up to the dose level of 1000 mg/kg body weight. Thus, after oral administration of LC for 90 days in rats, no observed adverse effect level (NOAEL) of LC was found to be 1000 mg/kg of body weight. A summary of adverse events is presented in [Table 14].
| Discussion|| |
LC is a combination of standardized extracts of seven herbs and genuine Saffron powder for treating sexual disorders, including mild-to-moderate ED and loss of libido. Almost all the ingredients used in LC possess aphrodisiac (libido enhancer), antistress, and antidepressant properties. A few ingredients of LC also help release nitric oxide from the nerve endings of the penis, which, in turn, helps relax the penile tissue and, thus, maintains an erection.,,,,,,,,,,, Also, in various research studies, these herbs are shown to help alleviate various elements involved in sexual dysfunction.,,,,,,,,,,,
For thousands of years, traditional Ayurvedic medicines have been used safely and effectively in managing various disease ailments. However, herbs used in traditional medicines are emerging with new extractive materials and advanced dosage forms. As per the demand of the scientific era, it is crucial to conduct toxicity studies to optimize the safety of plant-based medicines in consideration of their historical applications on humans. Hence, the present studies were designed to evaluate the safety of LC in experimental animals.,,,,,,,,,
In assessing and evaluating a test substance’s toxicity, determining the acute toxic effect by the oral route is one of the initial steps. Toxicity analysis is helpful for providing information on acute health hazards likely to arise from human overdoses. Acute toxicity data can serve as a base for the establishment of a safe dosing range for repeated administration and can provide basic information on side effects or toxic action of drug substances. Also, the conduct of acute toxicity studies in two rodent species (mice and rats) provides valid safety confirmation.,,,,,,,,,
In the principle of acute toxicity study, lethal dose50 calculation shows a proportion of death of the animals with expected lethality dose of the test drug. lethal dose50 value can be determined only when at least two doses result in mortality higher than 0% and lower than 100%. The predefined dosages of test substances (any) in acute findings improve the opportunity for laboratory-to-laboratory reporting consistency and repeatability.,,,,,,,,,
The repeated dose 90-day oral toxicity study provides evidences of potential health hazards, which can arise from repeated dosing exposure of test drug over a limited period. The data that originate in this type of study provides information on the detrimental effects of the drug on targeted organ tissues, the reversibility of toxic activity, and calculations for NOAEL. The findings of repeated dose subchronic oral toxicity studies can be used in selecting dose levels for chronic toxicity studies.,,,,,,,,,
In acute toxicity studies, LC at 2000 mg/kg dose level did not induce any sign of toxicity in experimental animals (rats and mice) throughout the studies. These observations indicated the nontoxicity of LC up to 2000 mg/kg as a single dose.
In the 90-day toxicity study, LC treatment group animals at and up to 1000 mg/kg did not show any death or abnormal sign compared with control group animals. This observation indicates the absence of toxicity of the LC up to this level in the study. In addition, the treated rats’ diets were well accepted, suggesting that the test item was not possibly causing any alteration in the food metabolism of animals. In microscopic findings, the animals of the treatment groups did not show any alteration in tissues compared with the control group. The statistical analysis revealed an insignificant difference in the weights of the adrenals, kidneys, testes/ovaries, liver, and heart at and up to 1000 mg/kg compared with concurrent controls.
Test item treatment did not show significant variation in hematology and clinical chemistry parameters at and up to 1000mg/kg dose level. Urinalysis did not reveal any substantial treatment-related toxicity. The incidence of necropsy findings was not dose-dependent and, hence, considered for toxicological assessment, which was confirmed on histopathological results at and up to 1000 mg/kg. Thus, in current toxicity studies, the nonappearance of drug-related toxicity signs is an indication of the harmless nature of the test drug (LC) over long-term exposure.
| Conclusion|| |
The study results established that the LC was nontoxic, up to 2000 mg/kg when administered as a single oral dose in SD rats and SA mice. Thus, LC can be classified as GHS “unclassified” or “category 5” for labeling requirement requisite for oral toxicity. NOAEL of LC can be considered 1000 mg/kg in experimental animals under the study conditions and dosages employed.
Financial support and sponsorship
Ari Healthcare Pvt. Ltd.
Conflicts of interest
There are no conflicts of interest.
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[Figure 1], [Figure 2], [Figure 3], [Figure 4], [Figure 5], [Figure 6], [Figure 7], [Figure 8], [Figure 9]
[Table 1], [Table 2], [Table 3], [Table 4], [Table 5], [Table 6], [Table 7], [Table 9], [Table 8], [Table 10], [Table 11], [Table 12], [Table 13], [Table 14]