1. INTRODUCTION:

According to the world health organization, 80% of the population in the developing country relies on herbal drugs for the treatment of various diseases. In the last couple of years, the use of herbal medicines has become very successful in the treatment of various types of illness^1,2. Undoubtedly, of around 1,500,000 plants researched, the vast majority of them contain toxic substances^3,4.Many people in our country use herbal drugs for self-medication; so, assessment of the efficacy and safety of the herbal medicines is becoming very necessary. Since very little information is accessible about the safety profile of the frequently used herbal drugs, along these lines, endeavors to explain the medical advantages and dangers of the natural medicines

have to be intensified and this need is of great importance to assess the intense and persistent toxic levels of the natural drugs under study⁵. Actually, there is the absence of evidence of the safety, quality, and efficacy of many herbal drugs. Toxicity is a declaration of being toxic, showing the condition of adverse effects due to the interactions between the poisonous substance (s) and the cell. Even though numerous herbal drugs are safe and non-harmful, numerous plants, right now utilized for the medications, have been confirmed to be strongly toxic as going through the results of their acute and sub-acute toxicity studies^6,7. Singara (Trapa natans L., Lythraceae family), an aquatic plant found from the warm temperature region of Asia and Africa, generally grows in the slow-moving water. From ancient times, this plant is being cultivated on large scale in India and China⁸. In the Indian Ayurvedic system of medicine, Trapa natans L. has been used in various chronic diseases and disorders related to liver, stomach, kidney, and so on. In different literatures, it has been reported that Trapa natans L is being used as astringent, somatic agent, febrifuge, and antiseptic⁹. Trapa natans L. fruits are used for the treatment of burning sensation, fatigue, inflammation, strangury, and fatigue¹⁰. Since very limited data has been obtained regarding the safety profile of this plant, it has become very necessary to evaluate the preclinical safety and toxicity profiles of the extract of the plant, Trapa natans L, for the determination of its future therapeutic potential. Therefore, the present study aims to determine the acute and sub-acute toxicities of the plant extract, for 14 days and 28 days, respectively, following the OECD guidelines 423 and 407, respectively^11,12.

2. MATERIALS AND METHODS:

2.1 Plant materials:

The Trapa natans L. plants were collected from the Purulia District, West Bengal, in the month of July 2018. Authentication of the plant was done by Dr. Anjula Pandey, Principle Scientist, National Bureau of Plant Genetics, New Delhi, India (Voucher No. NHCP/NBPGR/2013-7).

2.2 Animals:

Wistar albino rats (220 - 250g each) were obtained from the NIET Animal House, Greater Noida. All the animals were housed for 7 days before starting the toxicity study. All the animals were kept in the laboratory environment following OECD guidelines. In each cage, only 5 animals were allowed and each group was formed with 6 animals.

2.3 Extraction process:

The plant materials were shade dried at normal room temperature for a period of 15 days until a constant weight was attained. The air-dried plant materials were then crushed by using an electrical grinder. The powdered material was kept in an air-tight container prior to the extraction and then it was extracted using methanol in a Soxhlet apparatus at 70°C for 72 h. Finally, the solvent of the extract was evaporated in a rotary evaporator.

2.4 Determination of percentage yield of Trapa natans L:

The percentage yield of the extract was calculated using the following equation:

% Yield = (Weight of the extract obtained / Initial weight of the powdered drug taken) x 100

2.5 Acute Toxicity Study:

The acute toxicity study was approved by the Animal Ethical Committee (Protocol No. IAEC/NIET/2019/01/08) and performed following OECD guideline 423.

For the acute toxicity study, 30 Wistar albino female rats were taken and the animals were divided into five groups, each containing six animals. The group I, considered as the control group, received distilled water and no extract; the animals of group II, group III, group IV and group V received 300mg/kg, 1000mg/kg, 2000 mg/kg and 4000mg/kg Trapa natans L extract, respectively. After administration of the drug, the animals were kept under observation at 30 minutes interval for the first four hours and then twice a day for the next 14 days, after the administration of the drug.

2.6 Sub acute toxicity Study:

Subacute toxicity study was performed as per OECD guideline 407. The animals were divided into four groups, each containing 5 male and 5 female animals. The group I served as the control group and group II, group III, and group IV animals received Trapa natans L at 400mg/kg, 600mg/kg, and 1000mg/kg doses, respectively. The experiment was continued for the next 28 days¹³.

2.7 Hematological study:

After the last dosing, the animals were kept fasting overnight; only water was allowed ad libitum. The blood samples were collected after anesthetizing the experimental animals with the help of ketamine hydrochloride. The blood samples were collected by retro-orbital puncture with the help of microhematocrit tubes containing heparin and kept in blood sample containers containing potassium EDTA^14,15. For the hematological analysis of the collected blood samples, which included the analysis of hemoglobin, RBC, platelets, TLC, MCH, and MCV, the samples were sent to the Vet Serv Lab.

2.9 Biochemical analysis and histopathological study:

On the 29th day of the study, all the rats were anesthetized by using ketamine hydrochloride and sacrificed after overnight fasting. Blood samples were collected from all the test animals and kept in containers without EDTA, but containing only 11% w/v tri-sodium citrate^16,17 . The liver and kidney were isolated from the sacrificed animals and sent to the Vet serv Lab, Delhi, India, for histopathological study.

2.10 Statistical analysis:

All the data generated from the studies were expressed in terms of mean ± standard error mean. The experimental data were analyzed by the one-way ANOVA, using GraphPad prism 8 to determine the significance values. Test data were compared with that of the control and p-value 0.05 was considered as a measure for significance.

3. RESULTS AND DISCUSSION:

3.1 Acute toxicity:

In the acute toxicity study, no treatment related adverse effect or mortality was observed after oral administration of the plant extract in single dose of 300mg/kg, 1000 mg/kg, 2000mg/kg, and 4000mg/kg. Up to the 14^th day of observation, there was no notable change in body temperature, texture of skin and fur, eye color, food habit, frequency of urination, sign of sedation, as well as symptoms of diarrhea.

Consequently, considering all the parameters related to the safety profile of the plant extract upto the level of 4000 mg/kg, the drug was considered to be safe, as no treatment related death was observed and the LD50 value was assumed to be >4000mg/kg.

3.2 Sub-acute toxicity:

As observed from the results of the sub acute toxicity study, no sign of clinical toxicity had been observed in the plant extract treated groups at doses of 300, 600 and 1000mg/kg (oral), as compared to that of the control group. The results obtained from the hematological analysis of the collected blood samples showed no significant change in the hematological parameters observed with the experimental animals, as compared to that of the control group (Table 1).

Table 1: Hematological parameters in sub acute toxicity study.

Group	Control		*Test Animals Treated with T natans* L Extract**
	Control		400 mg/kg		600 mg/kg		1000 mg/kg
	Male	Female	Male	Female	Male	Female	Male	Female
Hb(g/dL)	14.5±0.5	15.2±0.4	13.6±0.8	15.98±0.7	12.5±0.9	15.12±0.7	12.5±2.	14.12±0.8
RBC (mill/mm³)	8.5±0.9	7.9±0.9	9.5±1.2	8.2±0.4	9.6±1.6	8.10±0.7	9.3±2.5	7.9±0.9
TLC (10³/mm)	10.5±2.2	9.5±1.2	11.2±2.1	10.8±1.2	10.7±1.2	9.23±1.5	11.9±2.1	10.12±1.8
MCV (fL)	55.87±3.63	57.55±2.51	60.15±4.12	59.67±5.17	58.17±2.87	61.87±5.12	53.58±2.95	58.86±3.89
MCH (pg)	15.7±1.62	18.7±1.15	15.5±1.47	16.3±1.02	15.5±1.47	16.3±1.02	18.5±0.97	19.3±1.92
Platelets (10³/mL)	634±18.56	795±23.86	625±12.50	656±33.3	706±11.55	615±23.52	686±21.5	620±16.14

Value expressed as mean ± standard error mean; n=5

Table 2: -Biochemical parameters in sub acute toxicity study.

Observation	Control Group		Test Animals Treated with T natans L Extract
	Control Group		400 mg/kg		600 mg/kg		1000 mg/kg
	Male	Female	Male	Female	Male	Female	Male	Female
ALT (IU/L)	48.5±1.8	45.5±2.3	42.23±1.5	40.5±1.3	40.2±1.5	41.5±2.3	43.25±1.3	39.5±2.3
AST (IU/L)	124.5±3.69	120.5±2.69	126.16±2.3	110.5±3.4	118.5±5.5	121.5±5.6	123.8± 2.5	124.8± 3.6
ALP (IU/L)	95.83±2.6	98.72±2.9	94.5±3.5	94.65 ±2.2	97.25±1.7	95.14 ±2.8	100.5±2.2	97.25 ±4.1
Albumin (g/L)	38.2±1.8	34.2±1.2	37.8±2.3	37.2±1.7	41±2.4	36.2±1.5	42±2.1	34.2±1.9
Globulin (g/L)	27.2±2.1	25.3±1.5	28.5±1.5	27.5±1.5	29.5±2.4	26.5±14	31±3.1	29.6±1.2
T. Protein (g/L)	65.4±4.1	67.2±2.1	70.2±2.5	66.5±1.5	64.1±1.8	62.3±14	71±4. 2	65.2±4.3
Bilirubin (mg/dL)	0.39±0.15	0.36±0.15	0.38± 0.25	0.39±0.15	0.47± 0.09	0.34±0.12	0.38± 0.05	0.37±0.15
Cholesterol (mg/dl)	125.5±5.41	130.5±4.2	129.5±3.9	133.5±4.5	126.5±4.7	118.5±5.7	124.5±6.2	119.5±5.1
LDL(mg/dl)	53.2±1.3	49.2±1.9	48.2±2.8	48.2±1.5	55.2±1.4	56.2±1.4	50.2±1.3	51.2±1.5
HDL(mg/d)	60.6±1.7	63.2±1.3	59.5±1.8	61.2±1.5	58.4±1.1	62.2±1.4	57 ±1.3	51.2±1.5
Triglyceride (mg/dL)	69.13±1.08	72.13±1.08	75.23±1.16	68.15±1.08	70.23±0.87	70.13±1.08	73.2 ±1.03	72.1±1.08
Creatinine (mg/dL)	0.74±1.2	0.82±1.4	0.60±0.9	0.74±1.2	0.78±0.5	0.69±2.1	0.54±1.1	0.84±1.2

Value expressed as mean± standard error mean; n=5;

The lipid profile analysis also showed no significant change in the observed experimental values, as compared to that of the control group.

Liver damage from any drug or any hepatotoxic agent is generally assessed by analysis of the plasma enzymes, such as aspartate amino transaminase (AST) and alanine amino trasaminase (ALT). The major changes in the serum marker enzyme indicate liver damage^18,19. The normal levels of total protein, albumin, globulin and bilirubin indicate the normal working of liver^20,21.

From the biochemical analysis, no significant change was observed in the levels of the biochemical enzymes. From the results of such analysis no significant difference was observed between the control and the test group (p>0.05) (Table 2). Histopathological study reports supported the biochemical analysis. From the histopathological study of the liver, no sign of liver damage and liver cell necrosis was observed at the maximum dose level of 1000mg/kg for 28 days^22,23. The histogram of the kidneys of the sacrificed test animals proved the extract of Trapa natans L. to be non-toxic and the safe for use (Figure 1)^24,25.

Figure 1: Isolated organs A: Kidney; B: Liver of Rat;C:Heart

Figure 2: A: Histograms of Liver; B: Histograms of Kidney

There was no abnormal change in weight of the organs, such as liver, kidney, spleen, heart, lungs and adrenal gland of the test animals, as compared to those of the control group (Table 3). The observed values proved the Trapa natans L. extract to be non-toxic.

Table 3: Effect of trapa natans L extract on organ weight of experimental rats in sub acute toxicity study.

Observation	Control Group	*Test Animals Treated with T natans* L Extract**
Observation	Control Group	400 mg/kg	600 mg/kg	1000 mg/kg
Liver (g)	8.14±0.17	9.05±0.13	7.98±0.25	8.56±0.34
Kidney (g)	2.04±0.04	1.95±0.034	1.96±0.015	2.12±0.017
Spleen (g)	0.54±0.05	0.604±0.12	0.59±0.02	0.63±0.05
Heart (g)	0.96±0.01	0.96±0.01	0.96±0.01	0.96±0.01
Lungs (g)	1.05±0.02	0.98±0.07	1.12±0.15	1.25±0.56
Adrenals (g)	0.06±0.01	0.05±0.02	0.06±0.01	0.05±0.01

Value expressed as mean ± standard error mean; n=6; the significance value p>0.05 as compared to the control

CONCLUSION:

From the experimental results, it can be concluded that the methanolic extract of the plant is safe and non-toxic up to the dose of 4000mg/kg. Toxicity study results suggest the LD50 value of the studied extract to be >4000mg/kg

ACKNOWLEDGEMENT:

The authors are thankful to Dr. Avijit Mazumder, Director of Noida Institute of Engineering Technology (Pharmacy Institute), Greater Noida, Uttar Pradesh, India, for providing all kind of support.

CONFLICT OF INTEREST:

There is no academic, financial, personal and commercial conflict of interest among the authors.

ABBREVIATION:

ALP: Alkaline phosphatase; AST: Aspartate transaminase; ALT: Alanine transaminase; HDL: High-density lipoprotein; LDL Low-density lipoprotein,

Hb: Haemoglobin; RBC: Red blood cell; TLC: Total leukocyte count; MCV: Mean corpuscular value; MCH: Mean Coupuscular Hemoglobin

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Received on 05.04.2021 Modified on 15.12.2021

Research J. Pharm. and Tech. 2022; 15(7):2923-2927.

DOI: 10.52711/0974-360X.2022.00487