Acute and subchronic toxicity profile of methanol extract of leaves of Fimbristylis miliacea (L.) Vahl

Abstract

Ethnopharmacological relevance

Fimbristylis miliacea (L.) Vahl (Cyperaceae) is a grass like herb habitually breeds as weed in paddy fields and mostly disseminated in tropical or sub-tropical countries of south and south-east Asia, northern Australia, and west Africa. The plant has been traditionally used to treat fever as a form of poultice. However, no scientific study regarding its toxicity profile has been testified.

Aim of the study

The study has been carried out to determine the potential toxicity of the methanol extract from leaves of the Fimbristylis miliacea, employing the technique of acute and subchronic oral administration in mice.

Materials and methods

In the acute toxicity study according to OECD guideline 425, oral administration of FM methanol extract at single doses of 2000 and 5000 mg/kg in both sexes of Swiss albino mice was performed. Toxic symptoms, abnormal behavior, changes in body weight, and mortality were observed for 14 consecutive days. In subchronic toxicity study according to OECD guideline 407, plant extract was administered orally at doses of 100, 500, 1000, and 2000 mg/kg daily for 28 days. The general toxic symptoms, abnormal behavior, changes in body weight were observed daily. Biochemical analysis of serum, and histopathological examination of liver were performed at the end of the study.

Results

No mortality, abnormal behavior and urination, changes in sleep, food intake, adverse effect, and non-linearity in body weight have been recorded during acute toxicity study at the doses of 2000 and 5000 mg/kg. Also, in subchronic toxicity study, FM extract produced no mortality or any kind of adverse effects in regards of general behavior, body weight, urination, sleeping routine, and food intake. In case of analysis of thirteen different biochemical parameters, concentrations of aspartate transaminase (AST) and glucose were altered significantly in male and female mice in both acute and subchronic study. Total cholesterol and triglycerides at 5000 mg/kg.bw were changed in male mice in acute toxicity study. On the other hand, female mice had altered triglycerides in subchronic test. All other critical parameters were found unaffected. In subchronic test, histopathological examination of liver demonstrated cellular necrosis at 2000 mg/kg.bw in both male and female mice while minor necrosis was observed at 1000 mg/kg.bw. Thus, the no observed adverse effect level (NOAEL) can be assumed around 1000 mg/kg.bw.

Conclusion

The present study suggests that treatment with FM extract does not reveal significant toxicity.

Graphical Abstract

Highlights

• •Acute and subchronic toxicity study showed no alterations in behavior of the experimental animals.
• •No mortality was identified throughout the study.
• •NOAEL is equivalent to 1000 mg/Kg body weight
• •All doses of plant extract showed significant decrease in serum glucose level in mice of both sexes.
• •Most of the critical biochemical and histopathological observations showed no significant alterations.

1. Introduction

Folklore medicines have been used to treat medical cases and improve health since long ago worldwide [1]. World Health Organization estimated that about 80 % of the population, in developing countries, prefer to use folklore remedies, especially plant extracts or remedies derived from them as their chief health remedy [2].

With the rising popularity of folklore medicines, scientific documentation of the safety, efficacy, adverse effects and toxicological profile of folklore medicines have become a matter of concern [1], [3]. Subsequently, to support this huge dependency on folklore traditional medicines and to discover new natural candidates as new ways to treat severe diseases, it has become a major concern to study the chemo-toxicology of medicinal plants [4], [5], [6]. Traditional medicines are widely used to treat severe diseases including fever, pain, diabetes, in some cases cancer, hepatitis, several skin and lever diseases etc. which also supports the statement that chemo-toxicological profile of these traditional folklore remedies should be revealed [7], [8].

Fimbristylis miliacea (L.) Vahl (Cyperacea), a grass-like herb habitually found extensively in paddy fields, as a weed comprises a fibrous root system. The plant belongs to the family Fimbristylis, also acknowledged as the genus of sedge. They are frequently found in damp lands of tropical and subtropical regions including Bangladesh, India, Nepal, Pakistan, Cambodia, Laos, Myanmar, Indonesia, Malaysia, Central America, West Africa, North Australia, etc. [9], [10]. Some of the species of Fimbristylis has been reported to have medicinal properties including Fimbristylis dichotoma with antimicrobial and antioxidant activity [11], Fimbristylis aphylla with antimicrobial, cytotoxic, and antidiarrheal activity [12], Fimbristylis littoralis having antioxidant activity [13].

The leaves of Fimbristylis miliacea is traditionally used to treat fever [14]. The investigating plant Fimbristylis miliacea has been reported to have significant antipyretic and anti-nociceptive activity [15], antioxidant and antidiarrheal activity [16], hypoglycemic activity [17] in an earlier study in our lab. Chemical characterization of this plant has already been reported, where it has been addressed that the plant contains, flavonoids, saponins, tannins, phenols, alkaloids, cardiac glycosides. And a higher amount of tannin content 215.72, phenolic content 154.13, flavanol 126.45 and flavonoid 83.14 mg/g of dry extract of FM [16]. But no detailed study on the toxicological profile of extract from leaves of Fimbristylis miliacea has been reported. Hence, with the aim of obtaining scientific information on its safety and possible toxicity, in continuation of our work, we have investigated acute and subchronic toxicity study of Fimbristylis miliacea in mice to ensure this plant has a safe toxicological profile to support the beneficial pharmacological properties as revealed in our previous studies.

2. Materials and methods

2.1. Collection, identification, and preparation of plant material

After collecting the whole plant of Fimbristylis miliacea [18] from the rural area of Manikgonj, Bangladesh (23° 52’ N, 89° 45’ E), it was authenticated by the expert taxonomist of Bangladesh National Herbarium (Voucher No.: DACB-46517). This plant grows like a weed and is not enlisted in the list of rare plants list no formal permission was required to collect this plant, but verbal permission was taken from the property owner before collecting the plant. Fresh leaves were then trimmed, cleaned, dried at room temperature, and finally powdered. About 250 gm of the powdered materials were then taken in a clean and flat-bottomed amber glass container to soak in 2.5 L of 80 % methanol at room temperature for fourteen days with occasional shaking and stirring. The solution was then filtered with a filter cloth and Whatman filter paper No. 1 and was concentrated with a rotary evaporator (RE-EV311-V, LabTech S.R.L, Italy) until it was concentrated to a brownish-black gummy concentrate which was designated as crude methanol extract, and it was then preserved at 4 ^oC till further use.

Percentage of extract yield was calculated by the following equation

$$\%\mathit{yield}=\frac{\mathit{Amount}\mathit{of}\mathit{extract}\mathit{yield}}{\mathit{Amount}\mathit{of}\mathit{plant}\mathit{material}\mathit{soaked}}x100\left(\%\right)$$

2.2. Selection and maintenance of animals

Healthy Swiss albino mice (23–26 g) were obtained at least one week before the experiments from the Animal Resource Center of International Centre for Diarrhoeal Disease Research, Bangladesh (ICDDR, B). All animals were maintained under standard environmental conditions (relative humidity 55–65 %, room temperature 25 ºC, and 12 h light-dark cycle) and had free access to standard rodent diet and water ad libitum. The experiment was done in the Animal Research Laboratory of our institution. The institutional animal ethics committee approved the experimental protocols (Slip no: 42/2020). All animals were handled with utmost care to minimize discomfort and pain. Besides, whole investigation was reported in accordance with ARRIVE guidelines [19], ensuring ethical compliance of scientific study involving animal.

2.3. Acute oral toxicity

An acute oral toxicity study was conducted according to the instructions of the Organization for Economic Cooperation and Development [20]. About 15 male and 15 female healthy Swiss Albino mice (weight: 23–26 gm) were used in this experiment. The animals were randomly divided into three groups according to their sex (n = 10; 5 males and 5 females per group). Group I (Control) received 1 % Tween 80 10 ml/kg (in distilled water) orally; Group II (Acute toxicity) received a dose of the FM extract of 2000 mg/kg in 1% Tween 80; Group III (Acute toxicity) received a limit dose of the FM extract of 5000 mg/kg in 1 % Tween 80.

Animals were then observed for mortality, signs of acute toxicity (bleeding, itching, urination, sweating, writhing, laxation), and behavioral changes (aggression, unusual vocalization, agitation, sedation and somnolence, convulsions, tremors, ataxia, catatonia, paralysis, fasciculation, prostration and unusual locomotion, abnormality in food consumption, and asphyxia) for the first 4 h, then every 2 h for 24 h and finally periodically up to 48 h. All experimental animals were individually observed daily for general behavior and body weight changes, severe toxicity symptoms, and mortality for 14 days after treatment. The LD₅₀ should be greater than 5 g/kg if three or more mice survived in each group per sex. At the end of the experimental period, all animals were weighed and sacrificed after anesthetizing using standard anesthesia (Isoflurane) for organ and blood collection following standard procedures.

2.4. Subchronic toxicity

Subchronic toxicity study was done according to the instructions of the Organization for Economic Cooperation and Development [21]. About 25 male and 25 female Swiss Albino mice weight around 23–26 gm were used in this experiment. The animals were randomly divided into five groups according to their sex (n = 10; 5 males and 5 females per group). Group I (Control) received 1 % Tween 80 10 ml/kg (in distilled water) orally; Group II received a dose of the FM extract of 100 mg/kg; Group III received a dose of the FM extract of 500 mg/kg, Group IV received a dose of the FM extract of 1000 mg/kg; and Group V received a dose of the FM extract of 2000 mg/kg. All doses were administered once daily for 28 days by oral gavaging.

After the first dose animals were observed for mortality, signs of toxicity, and behavioral changes (aggression, unusual vocalization, agitation, sedation and somnolence, convulsions, tremors, ataxia, catatonia, paralysis, fasciculation, prostration and unusual locomotion, abnormality in food consumption, and asphyxia) for the first 4 h, then every 2 h for 24 h and finally periodically up to 48 h. All experimental animals were individually observed daily for general behavior and body weight changes, toxicity symptoms, and mortality for 28 days after treatment. At the end of the experimental period, all animals were weighed and sacrificed after anesthetizing using standard anesthesia (Isoflurane 2 % v/v) for organ and blood collection following standard procedures.

2.5. Blood collection and serum preparation

All animals were anesthetized using standard anesthesia (Isoflurane, 2 % v/v) and blood was collected by direct cardiac puncture. Collected blood was then kept 30 min at 37 ^oC to be clotted which was then centrifuged at 2500 rpm at 4^o C for 10 min and serum was separated and preserved for biochemical analysis. All of the animals were sacrificed by decapitation later and the liver, kidney, brain, spleen, pancreas, heart, and lung were removed, weighed, cleaned in 0.9 % NaCl solution. [22], [23].

2.6. Relative organ and body weights study

The body weights were checked daily, and the organs were weighed using standard weighing balance to calculate relative organ weight on the day of sacrifice [24].

$\mathrm{Relative}\mathrm{organ}weight(\%)=\frac{\mathrm{Absolute}\mathrm{weight}\mathrm{of}\mathrm{organ}(\mathrm{g})}{\mathrm{weight}\mathrm{of}\mathrm{animal}\mathrm{on}\mathrm{sacrifice}day(\mathrm{g})}x100\%$

2.7. Biochemical analysis

Marker enzymes, for instance, alanine aminotransferase (ALT), alkaline phosphatase (ALP), aspartate aminotransferase (AST), total bilirubin (TB), total protein (TP), total cholesterol (TC), triglyceride (TG), high-density lipoprotein (HDL), low-density lipoprotein (LDL), glucose (Glu), albumin (Alb), creatinine (CR), uric acid (UA) were measured spectrophotometrically in serum samples employing commercially available HUMAN diagnostic kits (HUMAN Gesellschaft für Biochemica und Diagnostica mbH, Germany). [25], [26], [27].

2.8. Histopathological studies

The liver samples were sliced in a square shape, rinsed in 0.9 % saline, and fixed for 24 h in phosphate-buffered formalin (10 %) at pH 7. Small pieces were then transferred into tissue cassettes, embedded in paraffin wax, cut into a thickness of 3–4 µm, and stained with hematoxylin and eosin (H&E) by following the standard procedure [28]. Histopathological inspection of the slides was executed using a light microscope with 400x magnification. Histopathological examination was enthralled on toxic effects of the plant extract, such as inflammatory, necrotic, circulatory, and fibrotic lesions.

2.9. Statistical analysis

Statistical analysis was accomplished with One-way ANOVA followed by Tukey’s multiple comparisons test designed for comparison among multiple test groups using GraphPad Prism (version 9.0.0). Statistical significance of test results is presented: *p < 0.05, ^**p < 0.01, ^***p < 0.001 vs control group.

3. Results

3.1. Acute toxicity

3.1.1. General signs and mortality

No signs of behavioral changes and toxicity were observed after the administration of plant extracts. Food and water intake were normal after the administration of the plant extract. No deaths occurred within the first 48 h of observation and in the whole experiment time frame in both male and female animal groups, which confirms that in acute dosing the plant extract was safe. In the whole period of the study, no animals died revealing less toxicity of the plant extract. Both 2000 mg/kg and 5000 mg/kg body weight extract administration proved to be safe. The LD₅₀ should be greater than 5 g/kg as no morbidity and general toxicity symptoms were observed [29].

3.1.2. Body weight and relative organ weight

In 14 days acute toxicity study, treatment with two different doses of FM, the body weight of animals of both sexes increased at a linear relationship as compared with the control group (Fig. 1). However, 5000 mg/kg showed a slightly higher rate of increment in body weight. In case of relative organ weight analysis, only the ratio of kidney and body weight showed significant changes in male mice in both 2000 and 5000 mg/kg.bw. In female mice, on the other hand, only brain (at 2000 mg/kg.bw) and liver (at 5000 mg/kg.bw) weight ratio were significantly changed as compared with control group (Fig. 2).

Fig. 1.

Body Weight of male and female mice in acute toxicity study. ‘FM’ stands for Fimbristylis miliacea and 2000 and 5000 indicate doses of extract in mg/kg.bw. Statistical significance of test results is presented: *p < 0.05, ^**p < 0.01, ^***p < 0.001 vs control group.

Fig. 2.

Relative Organ Weight of male and female mice in acute toxicity study. ‘FM’ stands for Fimbristylis miliacea and 2000 and 5000 indicate doses of extract in mg/kg.bw. Statistical significance of test results is presented: *p < 0.05, ^**p < 0.01, ^***p < 0.001 vs control group.

3.1.3. Biochemical analysis

Serum biochemical analysis in acute toxicity study of Fimbristylis miliacea shows that serum level of markers enzymes didn’t change significantly in both male and female mice group as compared to control group except serum AST, ALP, TC, and TG. TC and TG concentrations were increased at 5000 mg/kg.bw. Serum ALP level decreased significantly as compared to control group in male mice. Total cholesterol and triglycerides levels increased significantly in male mice, but the increase was not significant in female mice. Serum glucose level decreased significantly for all doses as compared to control group in both male and female indicating hypoglycemic effect of the plant extract. All biochemical parameters are demonstrated in (Table 1 and Table 2).

Table 1.

Biochemical parameters of male mice acute toxicity study with F. miliacea extract.

Parameters	Control	Doses of Fimbristylis miliacea (mg/Kg B.W)
		FM2000	FM5000
ALT (U/L)	31.3 ± 1.3	53.6 ± 4.9	35.9 ± 3.1
AST (U/L)	39.7 ± 3.8	61.1 ± 8.5	159.7 ± 21.4*
ALP (U/L)	270.1 ± 20.1	207.5 ± 32.4	126.5 ± 16.4**
BIL (mg/dL)	3.08 ± 0.31	2.39 ± 0.21	3.6 ± 0.2
TC (mg/dl)	76.2 ± 9.5	148.7 ± 23.5	157.1 ± 21.8*
TG (mg/dl)	206.0 ± 33.2	294.8 ± 41.6	268.6 ± 35.4**
HDL (mg/dl)	24.2 ± 4.4	46.3 ± 8.0	54.3 ± 7.4
LDL (mg/dl)	10.8 ± 5.4	43.5 ± 12.3	49.1 ± 9.9
Glu (mg/dl)	57.4 ± 3.8	54.7 ± 2.8	38.5 ± 3.6*
Alb (g/L)	23.9 ± 1.4	25.7 ± 1.9	20.9 ± 1.2
TP (g/L)	85.3 ± 3.6	114.1 ± 4.1	96.7 ± 3.2
CR (mg/dl)	0.26 ± 0.02	0.28 ± 0.03	0.44 ± 0.03
UA (mg/dl)	3.59 ± 0.20	6.61 ± 0.86	4.53 ± 0.63
Hb (g/dl)	14.5 ± 0.6	14.8 ± 0.7	12.5 ± 0.3

‘FM’ stands for Fimbristylis miliacea and 2000 and 5000 indicate doses of extract in mg/kg.bw. Statistical significance of test results is presented: *p < 0.05, ^**p < 0.01, ^***p < 0.001 vs control group.

Table 2.

Biochemical parameters of female mice acute toxicity study with F. miliacea extract.

Parameters	Control	Doses of Fimbristylis miliacea (mg/Kg B.W)
		FM2000	FM5000
ALT (U/L)	32.2 ± 2.9	39.9 ± 7.9	35.8 ± 1.6
AST (U/L)	47.4 ± 5.7	52.9 ± 8.8	233.8 ± 24.9**
ALP (U/L)	168.7 ± 27.3	174.0 ± 18.8	142.4 ± 29.4
BIL (mg/dL)	2.03 ± 0.26	1.18 ± 0.15	1.49 ± 0.14
TC (mg/dl)	142.9 ± 36.4	122.5 ± 33.4	110.3 ± 17.9
TG (mg/dl)	246.3 ± 35.5	263.2 ± 26.3	349.1 ± 33.7
HDL (mg/dl)	42.3 ± 13.2	34.9 ± 13.7	27.9 ± 9.6
LDL (mg/dl)	51.5 ± 27.1	34.9 ± 14.8	12.6 ± 10.6
Glu (mg/dl)	70.1 ± 2.4	53.1 ± 0.9**	33.8 ± 2.7**
Alb (g/L)	27.3 ± 2.8	23.6 ± 1.9	24.2 ± 1.4
TP (g/L)	122.0 ± 5.3	114.9 ± 7.4	110.7 ± 3.6
CR (mg/dl)	0.29 ± 0.02	0.27 ± 0.02	0.46 ± 0.05
UA (mg/dl)	4.39 ± 0.90	5.79 ± 1.56	4.95 ± 0.97
Hb (g/dl)	11.9 ± 0.4	13.3 ± 0.2	10.6 ± 0.6

‘FM’ stands for Fimbristylis miliacea and 2000 and 5000 indicate doses of extract in mg/kg.bw. Statistical significance of test results is presented: *p < 0.05, ^**p < 0.01, ^***p < 0.001 vs control group.

3.2. Sub chronic toxicity

3.2.1. General signs and mortality

All groups were observed for two hours after administration of the four separate doses in both male and female mice groups on daily basis to identify signs and symptoms of toxicity or behavioral changes. No signs of behavioral changes and toxicity were observed after the administration of plant extracts in the subchronic toxicity study. Food and water intake were normal after the administration of the plant extract. No deaths occurred within the first 48 h of observation and in the whole experiment time frame in both male and female animal groups, which confirms that in subchronic dosing the plant extract was safe. In the whole period of the study, no animals died, revealing less toxicity of the plant extract. All doses of the plant extract administration proved to be safe.

3.2.2. Body weight and relative organ weight

In the subchronic toxicity study, 28 days treatment with four different doses of FM, the body weight of animals of both sexes increased at a linear relationship (increasing nature) in comparison with the control group (Fig. 3) in the routine follow up of 28 days. However, the male mice groups showed a higher rate of increasing body weight as compared to the female groups. Body weight at day 21 showed a significant increase in body weight of the male mice groups. The relative organ weight of female mice groups did not change significantly in comparison with the control group, except the relative weights of a few organs such as the liver and lung (Fig. 4). Male mice groups significant changes in relative organ weight for all groups, this is probably due to the elevated body weight and long-term study effect. The relative organ weight of the Spleen, pancreas, lung of male mice groups was increased significantly.

Fig. 3.

Body Weight of male and female mice in subchronic toxicity study. ‘FM’ stands for Fimbristylis miliacea and 100, 500, 1000 and 2000 indicate doses of extract in mg/kg.bw. Statistical significance of test results is presented: *p < 0.05, ^**p < 0.01, ^***p < 0.001 vs control group.

Fig. 4.

Relative Organ Weight ratio of male and female mice in subchronic toxicity study. ‘FM’ stands for Fimbristylis miliacea and 100, 500, 1000 and 2000 indicate doses of extract in mg/kg.bw. Statistical significance of test results is presented: *p < 0.05, ^**p < 0.01, ^***p < 0.001 vs control group.

3.2.3. Biochemical analysis

Serum biochemical analysis in subchronic toxicity study of Fimbristylis miliacea shows that serum level of markers enzymes didn’t change significantly in both male and female mice group as compared to the control group except serum AST level. The dose 1000 mg/kg showed more effective than the 2000 mg/kg dose both in male and female animals which can be the cause of the higher dose containing toxic chemicals at a higher rate and log-term exposure affects the physiology significantly. Concentration of ALP was also altered significantly in male mice at 5000 mg/kg.bw in acute study. Serum glucose level decreased significantly for all doses as compared to control group in both male and female indicating hypoglycemic effect of the plant extract which also supports the previous findings [17]. But the uric acid, creatinine, total cholesterol, triglycerides, LDL levels in both sexes decreased for all doses as compared to the control group which increased in the acute toxicity study. All biochemical parameters are demonstrated in (Table 3 and Table 4).

Table 3.

Biochemical parameters of male mice subchronic toxicity study with F. miliacea extract.

Parameters	Control	Doses of Fimbristylis miliacea (mg/Kg B.W)
		FM100	FM500	FM1000	FM2000
ALT (U/L)	48.5 ± 10.4	44.1 ± 2.6	41.5 ± 2.8	35.4 ± 1.4	31.3 ± 2.4
AST (U/L)	177.9 ± 14.2	273.6 ± 11.5*	267.9 ± 27.9	250.8 ± 7.2*	217.7 ± 9.5
ALP (U/L)	164.3 ± 7.5	110.1 ± 6.1	130.9 ± 11.1	140.8 ± 3.7	150.8 ± 9.4
BIL (mg/dL)	0.60 ± 0.24	0.88 ± 0.06	0.87 ± 0.14	0.65 ± 0.04	0.46 ± 0.08
TC (mg/dl)	143.2 ± 20.5	168.5 ± 3.8	159.9 ± 11.7	151.3 ± 3.9	146.2 ± 10.5
TG (mg/dl)	226.8 ± 20.1	228.6 ± 5.3	219.5 ± 20.7	208.7 ± 3.7	195.6 ± 15.2
HDL (mg/dl)	46.8 ± 6.5	62.9 ± 5.4	56.8 ± 5.8	53.5 ± 3.9	52.4 ± 5.1
LDL (mg/dl)	51.1 ± 22.9	59.9 ± 3.9	59.2 ± 9.7	56.0 ± 2.6	54.7 ± 5.0
Glu (mg/dl)	77.7 ± 3.2	63.8 ± 1.8	52.5 ± 1.3**	44.3 ± 2.1***	32.7 ± 2.1***
Alb (g/L)	25.7 ± 1.6	16.8 ± 1.7	21.8 ± 1.7	23.2 ± 0.9	25.4 ± 0.3
TP (g/L)	137.6 ± 17.9	120.7 ± 3.1	112.6 ± 2.1	107.2 ± 0.7	103.0 ± 1.5
CR (mg/dl)	0.53 ± 0.03	0.40 ± 0.02	0.50 ± 0.04	0.54 ± 0.02	0.58 ± 0.03
UA (mg/dl)	5.61 ± 0.74	5.95 ± 0.29	5.74 ± 0.51	5.10 ± 0.06	4.3 ± 0.4
Hb (g/dl)	15.4 ± 0.7	14.5 ± 0.8	14.3 ± 0.5	13.9 ± 0.8	11.3 ± 0.5

‘FM’ stands for Fimbristylis miliacea and 100, 500, 1000 and 2000 indicate doses of extract in mg/kg.bw. Statistical significance of test results is presented: *p < 0.05, ^**p < 0.01, ^***p < 0.001 vs control group.

Table 4.

Biochemical parameters of female mice subchronic toxicity study with F. miliacea extract.

Parameters	Control	Doses of Fimbristylis miliacea (mg/Kg B.W)
		FM100	FM500	FM1000	FM2000
ALT (U/L)	51.8 ± 4.9	40.3 ± 3.4	39.7 ± 2.3	36.9 ± 1.9	35.6 ± 1.9
AST (U/L)	158.4 ± 6.3	39.2 ± 2.3***	45.3 ± 2.4***	57.0 ± 6.6**	75.6 ± 6.7**
ALP (U/L)	216.6 ± 32.4	265.2 ± 5.1	252.4 ± 23.8	172.5 ± 18.8	141.9 ± 10.9
BIL (mg/dL)	1.10 ± 0.20	0.24 ± 0.02	0.37 ± 0.16	0.88 ± 0.06	1.2 ± 0.1
TC (mg/dl)	185.1 ± 48.0	159.1 ± 5.0	146.0 ± 7.9	108.7 ± 22.5	98.7 ± 4.5
TG (mg/dl)	268.1 ± 14.9	392.7 ± 6.5**	371.2 ± 21.3	275.5 ± 32.9	247.4 ± 16.9
HDL (mg/dl)	61.1 ± 18.0	58.1 ± 5.2	53.2 ± 5.0	38.9 ± 10.7	32.9 ± 3.2
LDL (mg/dl)	70.4 ± 30.4	22.5 ± 2.9	18.6 ± 7.7	14.7 ± 7.9	16.3 ± 5.0
Glu (mg/dl)	69.8 ± 3.5	67.8 ± 1.7	54.2 ± 2.1	46.7 ± 2.1	37.1 ± 1.6
Alb (g/L)	23.1 ± 3.4	17.1 ± 0.6	18.5 ± 0.8	19.0 ± 0.5	21.4 ± 0.4
TP (g/L)	86.5 ± 9.2	78.5 ± 2.9	70.7 ± 2.5	66.2 ± 1.4	62.3 ± 4.6
CR (mg/dl)	0.35 ± 0.02	0.3 ± 0.0	0.25 ± 0.01	0.2 ± 0.0	0.17 ± 0.02
UA (mg/dl)	7.73 ± 1.58	6.07 ± 0.07	5.24 ± 0.43	4.5 ± 0.4	4.29 ± 0.58
Hb (g/dl)	13.2 ± 0.6	12.4 ± 0.7	12.1 ± 0.6	11.5 ± 0.6	9.04 ± 0.64

‘FM’ stands for Fimbristylis miliacea and 100, 500, 1000 and 2000 indicate doses of extract in mg/kg.bw. Statistical significance of test results is presented: *p < 0.05, ^**p < 0.01, ^***p < 0.001 vs control group.

3.3. Histopathological observations in subchronic toxicity study

Histological observations showed intact cellular architecture of the liver in both male and female mice (Fig. 5). Groups treated with 2000 mg/kg.bw showed noticeable cellular necrosis in both male and female mice. In case of 1000 mg/kg.bw treated groups, minor cellular necrosis was observed. On the other hand, slightly deformed cellular architecture with no rupture was seen in 500 mg/kg.bw.

Fig. 5.

Histopathological observation subchronic toxicity study. A) Control (Male) Intact cellular architecture, B) FM100 (Male) Intact cellular architecture, C) FM500 (Male) Slightly deformed but no rupture or necrosis, D) FM1000 (Male) Minor cellular necrosis, E) FM2000 (Male) Cellular necrosis, F) Control (Female) Intact cellular architecture, G) FM100 (Female) Intact cellular architecture, H) FM500 (Female) Slightly deformed but no rupture or necrosis, I) FM1000 (Female) Minor necrosis, J) FM2000 (Female) Cellular necrosis. Magnification was 400x.

4. Discussion

When evaluating the toxicity level of a plant extract, mortality is a significant criterion as it shows the immediate result of severe toxicity [26]. Animal models are widely used to assess toxicity at the introductory level as the first and foremost identification of unfavorable effects can only be foretold in animal models easily without wasting massive resources and time. Several behavioral parameters have been observed in this study that reveals the toxicity level of the plant extract is tolerable [30], [31]. As all the mice groups didn’t show any sign of abnormal behavior after dose administration for the whole study period, it is clear that the plant extract has no severe toxicity in neurophysiology [32], [33]. The increase in both body weight and relative organ weight might indicate increased adiposity which can decrease the glucose level in the blood, also supports the previous findings [17]. The changes of biomarkers in acute toxicity study, that were statistically significant may be due to comparatively high doses exposure. An increase in AST level can occur due to changes in secondary metabolites and AST is considered a less specific marker for liver injury than ALT, as it is also expressed from other tissues, such as the brain, myocardial cells, and skeletal muscle cells [34]. A decrease in ALT and ALP level indicates the plant extract has a less toxic effect in the liver cells of the experimented animals [35]. In addition to this, damaged liver cells release increased amounts of ALP into the blood. ALP concentration in plasma also elevates with large bile duct obstruction, intrahepatic cholestasis, or infiltrative diseases of the liver. However, as the changes in the level of other biomarkers in serum weren’t statistically significant it is clear that the plant extract has a moderately less or no toxic effect, and it can be a potential candidate for a new natural drug source. Changes in biomarkers in subchronic toxicity study were less significant when analyzed statistically. However, these changes also indicate a less adverse effect of the plant extract on the mice physiology which ultimately indicates the investigating plant as a safe source of natural drug components. Natural compounds having negligible toxic effect are the preferable source of choice in search of new drug or therapeutic components [26]. Besides, scientific or experimented knowledge is a prerequisite for further investigation of specific therapeutic effects of medicinal plant, as this pre-study saves a huge number of time and resources [33]. Change in relative organ weight is a steadfast gauge that can be castoff in toxicological research to evaluate toxicity caused by chemicals present in a plant extract [36], [37].

In the overall study, the biochemical markers in both acute and subchronic toxicity study, didn’t alter significantly. Besides, body weight, relative organ weights, behavior, histopathological observations also supports the statement that the plant Fimbristylis miliacea exerts a safer chemical profile and indicates that the previous studies regarding the identification pharmacological properties of Fimbristylis miliacea done by [15], [16], [17] can be extended for further deep investigations to identify specific chemical compounds responsible for these effects.

5. Conclusion

The medicinal plant Fimbristylis miliacea possesses less toxic effect in both male and female rodent animals which is a positive indicator for the plant to be a safe and potential source of new drug candidate. To conclude with, the toxicological profile of the plant Fimbristylis miliacea does not alter the cellular, biomolecular, physiological ecosystem of the experimental animals hence it can be a potential source of new drug and further deep investigations is required to reveal the chemo-architecture of this plant.

Funding

The research work was partially funded by Institutional Research Cell.

Ethics approval and consent to participate

The Institutional Ethics Committee on Animal Experimentation approved all experimental protocols (Slip No.: 42/2020) and the experiments were performed according to state and institutional guidelines.

Consent for publication

All authors consent for publication of the manuscript.

CRediT authorship contribution statement

Roni Roy: Conceptualization, Methodology, Investigation, Formal analysis, Writing – original draft. Israt Jahan Liya: Investigation, Writing, Formal analysis. Jony Roy Investigation, Writing, Formal analysis. Mohammad Anwarul Basher: Conceptualization, Writing – review & editing, Supervision.

Declaration of Competing Interest

The authors declare the following financial interests/personal relationships which may be considered as potential competing interests: Roni Roy reports administrative support and equipment, drugs, or supplies were provided by Noakhali Science and Technology University.

Handling Editor: Prof. L.H. Lash

Appendix A. Supplementary material

Supplementary material.

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Supplementary material.

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Data availability

Supplementary data will be deposited to the journal and all data will be available on request.