Acute oral toxicity of Polyalthia longifolia var. pendula leaf extract in Wistar albino rats

Abstract

Context: Polyalthia longifolia (Sonn.) Thw. var. pendula (Annonaceae), a tall evergreen tree, is cultivated all over India. The plant is used in traditional systems of medicine for the treatment of fever, skin diseases, and hypertension.

Objective: The present study evaluated the acute oral toxicity of Polyalthia longifolia var. pendula leaf extract in Wistar albino rats.

Material and methods: The parameters evaluated daily after oral drug administration of the extract (540, 1080, 2160 and 3240 mg/kg body weight) were mortality, signs of toxicity, feed and water consumption and body weight changes up to 14 days. The effect of different doses of the extract on organ weight, biochemical and hematological parameters were evaluated on the 15th day.

Results and conclusion: Methanol extract of Polyalthia longifolia leaf up to the dose level 3240 mg/kg body weight did not produce any toxic effects or deaths; the extract was well tolerated by the rats. It did not alter body weight, feed and water consumption. The organ weight, biochemical and hematological analysis did not show any dose- dependant changes in any of the parameters examined in animals of both sexes. The acute oral administration of the methanol extract of Polyalthia longifolia leaf was not toxic and safe in a single dose.

Keywords::

• Single dose
• cage side observation
• relative organ weight
• hematology
• methanol extract

Introduction

The health promotive and disease preventive approach available in the Indian systems of medicine such as “Ayurveda” is gaining greater attention and popularity in many regions of the world. Medicinal plants from time immemorial have been used in virtually all cultures as a source of medicine (Cragg & Newman, 2001). They are considered to be the backbone of traditional medicine and are widely used to treat acute and chronic diseases. Research on pharmacognosy, chemistry, pharmacology, and clinical therapeutics have been carried out on ayurvedic medicinal plants and many of the major pharmaceutical corporations have changed their strategies in favor of natural products drug discovery (Waxler-Morrison, 1988). The World Health Organization estimated that perhaps 80% of the world’s inhabitants rely chiefly on traditional medicines. Therefore, WHO approved the use of herbal products for national policies and drug regulatory measures in order to strengthen research and evaluation of the safety and efficacy of these products. Farnsworth (1984) reported that of the 119 plant derived drugs listed in a WHO study, 74% were discovered as a result of chemical studies to isolate the active compounds responsible for the use of original plant in traditional medicine. The use of plants for healing purposes is gaining increasing popularity as they are believed to be beneficial and free of side effects. However, the rationale for the utilization of medicinal plants has rested largely on long-term clinical experience with little or no scientific data on their efficacy and safety (Zhu et al., 2002). Medicinal herbs have their use as medications based simply on traditional folk use that has been perpetuated along several generations. With the upsurge in the use of herbal medicines, a thorough scientific investigation of these plants is imperative, based on the need to validate their folkloric usage (Sofowora, 1989). Dosage is the most important factor influencing toxicity. A dose is the total amount of a substance administered to, taken or absorbed by an organism. Substances may accumulate over time to a dose that may be poisonous, or may enter the body in sufficiently high dose or concentration. Toxicological studies help to decide whether a new drug should be adopted for clinical use or not (Anisuzzaman et al., 2001). Depending on the duration of exposure of animals to drug, toxicological studies may be of three types viz. acute, sub-acute and chronic. In acute toxicity studies, a single dose of drug is given in large quantity to determine immediate toxic effect. Acute toxicity studies are commonly used to determine LD₅₀ of drug or chemicals, signs of toxicity and effects on biochemical and hematological parameters. In sub-acute toxicity studies, repeated doses of drug are given in sub-lethal quantity for a period of 14 to 21 days. Sub-acute toxicity studies are used to determine effect of drug on biochemical and hematological parameters of blood as well as to determine histopathological changes. In chronic toxicity studies, repeated doses of drug are given for a longer period ranging from 6 weeks to 3 months. The parameters studied are the same as that of sub-acute study.

Polyalthia longifolia (Sonn.) Thw. var. pendula (Annonaceae) is a tall evergreen tree cultivated all over India. The plant has been used in the traditional system of medicine for the treatment of fever, skin diseases, hypertension (Kirtikar & Basu, 1995), hepatoprotective, anti-inflammatory (Tanna et al., 2009), antimicrobial (Faizi et al., 2003; Murthy et al., 2005; Nair & Chanda, 2006), hypoglycemic and antihyperglycemic (Nair et al., 2007) and antiulcer activities (Malairajan et al., 2008). Polyalthia cerasoides (Roxb.) Bedd have potential antioxidant, cytotoxic and genotoxic activities (Ravikumar et al., 2008) and antiproliferative, apoptotic and antimutagenic activities (Ravikumar et al., 2010). The present work is an acute toxicological study of Polyalthia longifolia var. pendula leaf.

Materials and methods

Plant collection and extraction

The leaves of Polyalthia longifolia were collected from Rajkot, Gujarat, India in the year 2007. The plant was compared with voucher specimen (voucher specimen No. PSN4) deposited by Dr. P.S. Nagar at the Department of Biosciences, Saurashtra University, Rajkot, Gujarat, India. Fresh leaves were washed under running tap water, shade-dried, homogenized to fine powder and stored in air tight bottles. The powder was defatted in hexane and extracted in methanol for 24 h on a rotary shaker by the cold percolation method (Parekh & Chanda, 2007). The methanol extract was concentrated using a rotary vacuum to yield the solid mass. The yield obtained was 8.62%. The methanol extract was used for the entire study.

Animals

Wistar albino rats of both sexes were obtained from the animal house of Sarabhai Research Center (SRC), Ahmedabad. Animal colonies were maintained at the Department of Biosciences, Saurashtra University, Rajkot. Animals were fed with commercial rat and mouse food supplied by Pranav Agro Industries Ltd., Amrut Brand, Baroda and water ad libitum. They were maintained in a 12 h light/dark cycle at 25 ± 2°C. The study was approved by CPCSEA (Committee for the Purpose of Control and Supervision on Experiments on Animals, Reg. No. 757/03/a/CPCSEA).

Selection of the dose

The dose considered for the experiment on rats was obtained from conversion of human dose of Polyalthia longifolia (3 g/kg). The conversion factor of human dose (per 200 g body weight) is 0.018 for rats (Ghosh, 1984). Hence, the calculated dose for the rats is 270 mg/kg. Thus, acute toxicity was done at four different doses, 540, 1080, 2160 and 3240 mg/kg body weight.

Acute toxicity studies

The acute oral toxicity (Ryu et al., 2004), study was performed as per OECD-404 guidelines (OECD, 1987); 10 rats/group (5 males and 5 females) were used for the study. Group 1 served as control and the other four groups were treated with extracts at different doses (540, 1080, 2160 and 3240 mg/kg body weight). A single dose of the extract was administrated orally to each animal. Signs of toxicity, body weight and feed and water consumption of each animal were observed every day for 14 days.

Cage side observation

In single oral dose toxicity testing, the animals were observed prior to dosing. Thereafter, observations were made at every hour for 5 h, then at 24 h, and then every day for 14 days. All observations were systematically recorded, with individual records being maintained for each animal. Cage side observations included evaluation of skin and fur; eyes; respiratory effect; autonomic effects, such as salivation, diarrhea, urination; and central nervous system effects, including tremors and convulsions, straub tail, ptosis, relaxation, changes in the level of activity, gait and posture, reactivity to handling, altered strength and stereotypy (Demma et al., 2007; Nair et al., 2009).

Feed and water consumption and body weight measurement

During the entire study period (14 days), animals were monitored daily for mortality, feed and water consumption, and any changes in body weight. Feed intake was calculated as g/animal/day. Water intake was calculated as ml/animal/day. Individual animal body weights were recorded daily until the end of the experiment.

Biochemical analysis

At the end of the study, all animals were fasted for 12 h and then, under mild ether anesthesia, animals were sacrificed and blood samples were collected. The biochemical parameters viz. total protein, albumin, urea (Gornall et al., 1949; Fawcett & Scott, 1960; Corcoran & Durnan, 1977), cholesterol, alkaline phosphatase (ALP), glutamate oxaloacetate transaminase (GOT), and glutamate pyruvate transaminase (GPT) (Kind & King, 1954; Reitman & Frankel, 1957; Trinder, 1969) were estimated from serum using kits (Span Diagnostic Ltd., Sachin, Gujarat, India). The absorbance of all the biochemical parameters was measured in a UV-VIS Spectrophotometer (Shimadzu, Tokyo, Japan).

Hematological analysis

At the end of the study, all animals were fasted for 12 h and then, under mild ether anesthesia, animals were sacrificed and blood samples collected. Blood was collected immediately into tubes containing EDTA for analysis of hematological parameters viz. hemoglobin, total red blood cells (R.B.C.), packed cell volume, mean cell volume (M.C.V.), mean cell hemoglobin (M.C.H.), mean cell hemoglobin concentration (M.C.H.C.), total white blood cells (W.B.C.), neutrophils, lymphocytes, eosinophils, monocytes, basophiles, total platelet count (Theml et al., 2004) using a hematology analyzer Sysmex XS800i (Sysmex Corporation, Holliston, Massachusetts, USA).

Organ weight analysis

Thymus glands, lungs, heart, liver, spleen, kidneys, adrenal glands, testes, uterus and ovaries were dried with filter paper, weighed using a Sartorius analytic balance A120S (Germany) and the relative weights calculated and expressed as g/100 g b.w.

Statistical analysis

The results are expressed as mean ± SEM (n = 5); data was analyzed using Student’s t-test and results are considered significant when p < 0.05.

Results

Cage side observation

Normal behavior with no mortality was observed in animals of the both sexes at all dose levels of methanol extract of P. longifolia during the entire experimental period.

Feed and water consumption and body weight measurement

Feed and water consumption pattern during the 14 days treatment period did not show dose-dependent and time related decline (Figure 1a, 1b and 2a, 2b). The amount of feed and water consumption of dosed groups was not significantly (p < 0.05) different from that of control group. Feed and water consumption was normal during the entire study in animals of both sexes at all dose levels.

Figure 1.  (a) Changes in feed consumption of male rats in acute toxicity study. (b) Changes in feed consumption of female rats in acute toxicity study.

Figure 2.  (a) Changes in water consumption of male rats in the acute toxicity study. (b) Changes in water consumption of female rats in the acute toxicity study.

There were no significant (p < 0.05) changes in the body weight of rats of both sexes from day 0 to day 14 in all the groups. Body weight of dosed and control rats did not show any significant (p < 0.05) change throughout the duration of treatment (Figure 3a and 3b).

Figure 3.  (a) Changes in body weight of male rats in the acute toxicity study. (b) Changes in body weight of female rats in the acute toxicity study.

Biochemical analysis

The effect of single dose oral administration of the methanol extract of P. longifolia on the serum biochemical parameters is shown in Tables 1 and 2. The treated male rats showed significant (p < 0.05) differences from control groups in total protein (1080 mg/kg, 2160 mg/kg p < 0.05), albumin (3240 mg/kg, p < 0.05), urea (540 mg/kg, 1080 mg/kg, 2160 mg/kg, 3240 mg/kg, p < 0.05), ALP (1080 mg/kg, 3240 mg/kg), GOT (1080 mg/kg, 2160 mg/kg, p < 0.05), and GPT (540 mg/kg, 1080 mg/kg, 2160 mg/kg, 3240 mg/kg, p < 0.05). The treated female rats showed significant (p < 0.05) differences from control in urea (3240 mg/kg, p < 0.05), cholesterol (540 mg/kg, 2160 mg/kg, p < 0.05), GOT (540 mg/kg, p < 0.05), and GPT (540 mg/kg, 2160 mg/kg, 3240 mg/kg, p < 0.05).

Table 1.  Effect of single oral administration of methanolic extract of P. longifolia on biochemical parameters of male rats.

Group	Total protein (g/dl)	Albumin (g/dl)	Urea (mg/dl)	Cholesterol (mg/dl)	ALP KA Units	GOT (IU/L)	GPT (IU/L)
Control	6.48 ± 0.04	4.38 ± 0.02	68.22 ± 3.34	89.55 ± 2.73	71.38 ± 7.99	90.20 ± 2.85	21.40 ± 0.60
540 mg/kg	6.60 ± 0.06^ns	4.64 ±0.17^ns	46.16 ± 1.07*	78.34 ± 2.95^ns	56.88 ± 7.16^ns	108 ± 9.27^ns	26.20 ± 0.37*
1080 mg/kg	6.71 ± 0.05*	4.19 ± 0.08^ns	44.82 ± 2.22*	105.14 ± 3.03^ns	47.65 ± 2.07*	57.40 ± 11.2*	15.60 ± 3.19*
2160 mg/kg	6.76 ± 0.07*	4.60 ± 0.10^ns	49.66 ± 0.77*	88.43 ± 2.73^ns	58.42 ± 3.97^ns	108.60 ± 3.84*	37.80 ± 1.66*
3240 mg/kg	5.65 ± 0.38^ns	3.87 ± 0.17*	38.86 ± 2.16*	112.29 ± 5.43^ns	44.61 ± 2.14*	85.60 ± 1.03^ns	13.00 ± 1.76*

Values are expressed as mean ± SEM, n = 5, *p < 0.05 when compared with Control using Student’s t-test.

ns, not significant.

Table 2.  Effect of single oral administration of methanolic extract of P. longifolia on biochemical parameters of female rats.

Group	Total protein (g/dl)	Albumin (g/dl)	Urea (mg/dl)	Cholesterol (mg/dl)	ALP KA Units	GOT (IU/L)	GPT (IU/L)
Control	7.05 ± 0.11	4.90 ± 0.10	56.36 ± 2.07	97.04 ± 6.07	27.54 ± 4.39	98.60 ± 3.91	21.80 ± 0.66
540 mg/kg	7.03 ± 0.16^ns	4.76 ± 0.26^ns	52.09 ± 1.33^ns	76.68 ± 1.77*	35.69 ± 5.17^ns	128.20 ± 9.32*	34.20 ± 1.59*
1080 mg/kg	7.16 ± 0.06^ns	4.84 ± 0.08^ns	54.85 ± 3.77^ns	98.97 ± 4.33^ns	28.27 ± 5.24^ns	99.60 ± 5.55^ns	20.80 ± 0.58^ns
2160 mg/kg	6.91 ± 0.10^ns	5.04 ± 0.10^ns	52.17 ± 0.99^ns	70.69 ± 1.86*	31.45 ± 4.95^ns	105.40 ± 6.75^ns	29.80 ± 1.62*
3240 mg/kg	6.96 ± 0.10^ns	4.60 ± 0.15^ns	42.96 ± 2.16*	98.24 ± 1.36^ns	18.66 ± 2.49^ns	100.80 ± 10.49^ns	10.80 ± 1.02*

Values are expressed as mean ± SEM, n = 5, *p < 0.05 when compared with Control using Student’s t-test.

ns, not significant.

Hematological analysis

The effects of single dose oral administration of the methanol extract of P. longifolia on the hematological parameters are shown in Tables 3 and 4. The treated male rats showed significant (p < 0.05) differences from control groups in RBC counts (540 mg/kg, 2160 mg/kg, p < 0.05), PCV (540 mg/kg, 2160 mg/kg, p < 0.05), MCH (540 mg/kg, 1080 mg/kg, 2160 mg/kg, p < 0.05), MCHC (540 mg/kg, 2160 mg/kg, 3240 mg/kg, p < 0.05), lymphocytes (540 mg/kg, p < 0.05), and eosinophils (540 mg/kg, p < 0.05). The treated female rats showed significant differences from control groups in hemoglobin (2160 mg/kg, p < 0.05), PCV (2160 mg/kg, p < 0.05), MCH (540 mg/kg, 2160 mg/kg, p < 0.05), MCHC (540 mg/kg, 2160 mg/kg, p < 0.05), and total platelet count (540 mg/kg, p < 0.05). However, there was no significant change in the levels of total WBC counts, neutrophils, monocytes, and basophiles at all doses.

Table 3.  Effect of single oral administration of methanolic extract of P. longifolia on hematological parameters of male rats.

Group	Control	540 mg/kg	1080 mg/kg	2160 mg/kg	3240 mg/kg
Hemoglobin (g/dl)	14.82 ± 0.17	15.48 ± 0.32^ns	14.94 ± 0.47^ns	15.40 ± 0.35^ns	15.22 ± 0.32^ns
Total RBC (ml/cu.mm)	7.91 ± 0.09	8.57 ± 0.19*	7.80 ± 0.28^ns	8.48 ± 0.23*	8.16 ± 0.09^ns
Packed Cell Volume (%)	41.02 ± 0.68	44.48 ± 0.97*	41.02 ± 1.65^ns	45.04 ± 1.00*	41.10 ± 0.61^ns
MCV (cu.micron)	52.08 ± 0.33	51.9 ± 0.43^ns	52.57 ± 0.67^ns	53.12 ± 0.66^ns	50.38 ± 0.78^ns
MCH (pico gram)	18.74 ± 0.08	18.08 ± 0.20*	19.17 ± 0.14*	18.16 ± 0.20*	18.66 ± 0.38^ns
MCHC (%)	35.98 ± 0.23	34.82 ± 0.28*	36.48 ± 0.43^ns	34.2 ± 0.20*	37.02 ± 0.34*
Total WBC/10^3 mm^3	6.8 ± 0.79	7.66 ± 1.06^ns	10.0± 1.9^ns	6.9 ± 1.02^ns	10.8 ± 1.61^ns
Neutrophils (%)	20.6 ± 1.75	12.8 ± 3.77^ns	22.60 ± 1.66^ns	25.6 ± 5.08^ns	31.20 ± 5.30^ns
Lymphocytes (%)	78 ± 1.84	88.4 ± 3.54*	76.80 ± 1.96^ns	73.8 ± 5.34^ns	67.80 ± 5.48^ns
Eosinophils (%)	1.4 ± 0.24	0*	0.60 ± 0.40^ns	0.6 ± 0.40^ns	1 ± 0.32^ns
Monocytes (%)	0	0^ns	0^ns	0^ns	0^ns
Basophiles (%)	0	0^ns	0^ns	0^ns	0^ns
Total platelet count/10^3 mm^3	126.8 ± 10.8	123.7 ± 10.0^ns	141.2 ± 7.2^ns	90.5 ± 16.5^ns	120.3 ± 12.2^ns

Values are expressed as mean ± SEM, n = 5, *p < 0.05 when compared with Control using Student’s t-test.

ns, not significant; RBC, red blood cell; MCV, mean corpuscular volume; MCH, mean corpuscular hemoglobin; MCHC, mean corpuscular hemoglobin concentration; WBC, white blood cell.

Table 4.  Effect of single oral administration of methanolic extract of P. longifolia on hematological parameters of female rats.

Group	Control	540 mg/kg	1080 mg/kg	2160 mg/kg	3240 mg/kg
Hemoglobin (g/dl)	15.74 ± 0.17	15.24 ± 0.25^ns	15.38 ± 0.34^ns	15.08 ± 0.09*	15.52 ± 0.24^ns
Total RBC (ml/cu.mm)	7.89 ± 0.05	8.28 ± 0.20^ns	7.73 ± 0.28^ns	8.22 ± 0.13^ns	8.01 ± 0.18^ns
Packed cell volume (%)	41.08 ± 0.47	42.66 ± 0.99^ns	38.26 ± 2.74^ns	42.8 ± 0.29*	41.06 ± 1.07^ns
MCV (cu.micron)	52.04 ± 0.41	51.22 ± 0.22^ns	49.35 ± 2.60^ns	52.1 ± 0.61^ns	51.29 ± 0.27^ns
MCH (pico gram)	19.94 ± 0.14	18.4 ± 0.18*	19.94 ± 0.33^ns	18.36 ± 0.28*	19.41 ± 0.29^ns
MCHC (%)	38.32 ± 0.07	35.74 ± 0.27*	41.04 ± 3.10^ns	35.22 ± 0.20*	37.84 ± 0.44^ns
Total WBC/10^3 mm^3	7.60 ± 0.70	8.3 ± 1.44^ns	9.0± 0.98^ns	5.9 ± 0.79^ns	10.0 ± 1.67^ns
Neutrophils (%)	22.8 ± 3.87	24.2 ± 3.65^ns	19.60 ± 2.09^ns	15.6 ± 1.36^ns	26.20 ± 4.07^ns
Lymphocytes (%)	77 ± 3.99	75.8 ± 3.65^ns	80 ± 2.28^ns	84.4 ± 1.36^ns	73.20 ± 4.40^ns
Eosinophils (%)	0.20 ± 0.20	0^ns	0.40 ± 0.40^ns	0^ns	0.60 ± 0.40^ns
Monocytes (%)	0	0^ns	0^ns	0^ns	0^ns
Basophiles (%)	0	0^ns	0^ns	0^ns	0^ns
Total platelet count/10^3 mm^3	119.6 ± 9.8	77.7 ± 9.1*	123.0 ± 8.5^ns	151.7 ± 6.7^ns	106.2 ± 18.3^ns

Values are expressed as mean ± SEM, n = 5, *p < 0.05 when compared with Control using Student’s t-test.

Ns, not significant; RBC, red blood cell; MCV, mean corpuscular volume; MCH, mean corpuscular hemoglobin; MCHC, mean corpuscular hemoglobin concentration; WBC, white blood cell.

Relative organ weight

The effect of single dose oral administration of the methanol extract of P. longifolia on the relative organ weights is shown in Tables 5 and 6. The treated male rats showed significant differences from control groups in liver (2160 mg/kg, p < 0.05), spleen (540 mg/kg, 2160 mg/kg, p < 0.05), kidneys (3240 mg/kg, p < 0.05), adrenal glands (3240 mg/kg, p < 0.05) and testes (540 mg/kg, 3240 mg/kg, p < 0.05). However, there was no significant change in the relative weights of lungs, heart and thymus glands at any dose levels of treated male rats as compare to control. The treated female rats showed significant differences from control in heart (2160 mg/kg, p < 0.05), liver (2160 mg/kg, p < 0.05) and adrenal glands (2160 mg/kg,p < 0.05). However there was no significant change in relative weight of lungs, spleen, kidneys, thymus glands and uterus and ovaries at any dose levels of treated female rats as compared to control group.

Table 5.  Effect of single oral administration of methanolic extract of P. longifolia on relative organ weights of male rats (g/100 g of body weights).

Group	Thymus glands	Lungs	Heart	Liver	Spleen	Kidneys		Adrenal glands		Testes
						Left	Right	Left	Right	Left	Right
Control	0.25 ± 0.02	0.69 ± 0.07	0.32 ± 0.00	2.65 ± 0.01	0.25 ± 0.02	0.37 ± 0.01	0.37 ± 0.02	0.009 ± 0.001	0.011 ± 0.002	0.48 ± 0.01	0.48 ± 0.01
540mg/kg	0.29 ± 0.03	0.61 ± 0.02	0.32 ± 0.01	2.55 ± 0.11	0.19 ± 0.01*	0.33 ± 0.01	0.34 ± 0.01	0.012 ± 0.002	0.011 ± 0.001	0.51 ± 0.01	0.52 ± 0.01*
1080 mg/kg	0.23 ± 0.02	0.64 ± 0.06	0.31 ± 0.01	2.71 ± 0.05	0.22 ± 0.01	0.34 ± 0.01	0.34 ± 0.01	0.010 ± 0.009	0.010 ± 0.009	0.45 ± 0.02	0.48 ± 0.01
2160 mg/kg	0.21 ± 0.02	0.57 ± 0.02	0.31 ± 0.00	2.30 ± 0.05*	0.20 ± 0.01*	0.36 ± 0.01	0.36 ± 0.01	0.010 ± 0.001	0.008 ± 0.001	0.48 ± 0.02	0.48 ± 0.02
3240 mg/kg	0.28 ± 0.03	0.74 ± 0.02	0.35 ± 0.02	2.79 ± 0.07	0.27 ± 0.02	0.41 ± 0.01*	0.42 ± 0.01	0.014 ± 0.001*	0.010 ± 0.001	0.53 ± 0.01*	0.50 ± 0.02

Values are expressed as mean ± SEM, n = 5, *p < 0.05 when compared with Control using Student’s t-test.

Table 6.  Effect of single oral administration of methanolic extract of P. longifolia on relative organ weights of female rats (g/100 g of body weights).

Group	Thymus glands	Lungs	Heart	Liver	Spleen	Kidneys		Adrenal gland		Uterus and Ovaries
						Left	Right	Left	Rights
Control	0.31 ± 0.02	0.83 ± 0.06	0.33 ± 0.01	2.75 ± 0.05	0.27 ± 0.03	0.37 ± 0.02	0.37 ± 0.02	0.018 ± 0.002	0.015 ± 0.002	0.35 ± 0.02
540 mg/kg	0.33 ± 0.05	0.67 ± 0.03	0.31 ± 0.01	2.55 ± 0.10	0.26 ± 0.00	0.33 ± 0.01	0.33 ± 0.01	0.017 ± 0.002	0.018 ± 0.002	0.33 ± 0.02
1080 mg/kg	0.29 ± 0.03	0.76 ± 0.03	0.32 ± 0.01	2.66 ± 0.04	0.26 ± 0.01	0.37 ± 0.01	0.35 ± 0.01	0.014 ± 0.002	0.015 ± 0.001	0.38 ± 0.03
2160 mg/kg	0.29 ± 0.03	0.72 ± 0.06	0.30 ± 0.00*	2.46 ± 0.05*	0.24 ± 0.01	0.36 ± 0.02	0.35 ± 0.02	0.013 ± 0.001*	0.019 ± 0.003	0.31 ± 0.02
3240 mg/kg	0.29 ± 0.03	0.85 ± 0.06	0.32 ± 0.01	2.74 ± 0.11	0.26 ± 0.01	0.37 ± 0.01	0.36 ± 0.01	0.018 ± 0.002	0.022 ± 0.002	0.33 ± 0.02

Values are expressed as mean ± SEM, n = 5, *p < 0.05 when compared with Control using Student’s t-test.

Discussion

Herbal medicines are used worldwide for the treatment and prevention of various acute and chronic diseases and are gaining popularity in developing countries. Herbal medicines are often believed to be harmless because they are “natural” and are easily available and commonly used for self-medication without supervision. This increase in popularity and the scarcity of scientific studies on their safety and efficacy have raised concerns regarding toxicity and adverse effects of these medicines (Saad et al., 2006). These medicines contain bioactive constituents with potential to cause adverse effects (Bent & Ko, 2004).

The results of the present study suggested that Polyalthia longifolia is a relatively nontoxic plant. According to Nair et al. (2009) there were no gross behavioral changes in mice fed with various solvent extracts of P. longifolia and suggested that this plant is safe in acute study. The determination of feed and water consumption are important in the study of safety of a natural product, as proper intake of feed and water are necessary to the physiological status of the animals and to the achievement of the proper response to the drug tested instead of a “false” response due to improper nutritional conditions (Stevens & Mylecraine, 1994). During 14 days period of acute toxicity evaluation, rats were not associated with any mortalities and abnormalities in general conditions, behavior and growth, feed and water consumption of animals. Body weight changes are indicators of adverse side effects, as the animals that survive cannot lose more than 10% of the initial body weight (Teo et al., 2002). Body weight gain and feed consumption levels were similar in both control and treated animals.

The body weight and feed consumption was not affected by administration suggesting that methanol extract of P. longifolia did not induce appetite suppression and had no deleterious effect on health status, growth or development of animals.

Total protein measurement is used in the diagnosis of a variety of diseases involving the liver or kidney as well as other metabolic disorders. A decrease in albumin level has been attributed to several causes, such as massive necrosis of the liver, deterioration of liver function, hepatic resistance to insulin and glycogen impairment of oxidative phosphorylation (Rao, 1995). The total protein level increased in treated animals which demonstrate that the methanol extract of P. longifolia has no toxic effect on liver and kidney function. Albumin levels showed a similar trend but the increased levels were not significant. Urea and creatinine are compounds derived from proteins which are eliminated by the kidney. In the present work, accumulation of urea, a relevant indicator for renal impairment (Vidal et al., 2003) was not observed. In fact, the urea level significantly decreased at all dose levels in male rats while in female rats the levels decreased but were significant only at highest dose. The decrease in cholesterol levels in the treated animals showed that the methanol extract of P. longifolia has hypolipidemic activity and potential against cardiovascular risk factor (Zhou et al., 2004).

ALP, GOT, GPT in tissue and blood are important marker enzymes which are used to assess the integrity of the cell membrane, cytosolic activity and cell death (Akanji et al., 1993). The extent of hepatocellular injury is assessed by the increased serum levels of ALP, GOT and GPT. These damages could be acute or chronic, reversible or irreversible (Janbaz et al., 2002). In the present study, ALP levels decreased significantly by administration of methanol extract of P. longifolia which suggest that no possible cholestasis occurred at the dose levels tested since a rise in ALP level is usually a characteristic finding in cholestatic liver disease (Aniagu et al., 2005).

In male rats, GOT levels increased significantly only at dose level 2160 mg/kg b.w. and in female rats at dose level 540 mg/kg b.w. In both sexes, at other dose levels, GOT levels decreased or slightly increased but they were insignificant. GPT levels in both sexes, at some dose level, showed increased activity, although the extract showed a significant decrease at the highest dose in both male and female rats. All the three serum marker enzymes showed decreased levels except in some which were not dose dependent hence it can be stated that methanol extract of P. longifolia is hepatoprotective in nature.

The fall in hemoglobin content, RBC count and PCV can be correlated with induction of anemia, defective hematopoiesis, weakness and morbidity in experimental rats (Criswell et al., 2000). The present study showed that there was an increase in hemoglobin content and RBC count. Also it showed an increase in PCV levels which suggests that the extract had no allergic response and there was no risk of such diseases. The methanol extract has some polar constituent which may alter the amount of hemoglobin and RBCs in the blood. This is similar to the results obtained with some other plants (Sanchez-Elsner et al., 2004; Mbaka et al., 2010).

The increase in MCV and decrease in MCHC indicate macrocytic and hypochromic anemia (Barger, 2003). The study showed that there was no significant difference in MCV at any dose levels of animals of both sexes. The MCHC significantly decreased at two intermediate doses except at high dose in male rats but it was unaffected in female rats.

WBC and its subpopulations relating to it such as lymphocytes usually show increase in activity in response to toxic environment (Robins, 1974). In this study, WBC was not significantly altered, while lymphocytes, the main effectors cells of the immune system (Mc Knight et al., 1999), showed significant increase at low dose level of male rats. However, at other doses, male rats showed decreased levels of lymphocytes thus suggesting that the methanol extract of P. longifolia has no risk on the immune system of the animals.

Eosinophils normally constitute up to 7% of total circulating leukocytes. Eosinophils are important in the phagocytosis of foreign bodies. Eosinophils are also involved in allergic reactions (Oyesanmi et al., 1999). Eosinophils level usually show increases in allergic reactions, but this study showed that the eosinophils decreased at all the dose levels in male rats and at two intermediate doses in female rats. So it can be stated that animals have no allergic reaction when methanol extract of P. longifolia is administered orally.

In the present study, the level of platelets decreased significantly in female rats at low dose. The treated male rats showed non-significant decrease in the levels of platelets. Platelets, also known as thrombocytes, help to mediate blood clotting, which is a meshwork of fibrin fibers. The fibers also adhere to damaged blood vessels; therefore, the blood clot becomes adherent to any vascular opening and thus prevents further blood clot (Andrews et al., 1997). The extract could thus precipitate thrombocytopaenia which is the presence of low level platelets in the circulatory system. If someone suffers from thrombocytopaenia, there is a tendency to bleed (Body, 1996). This observation of decreased platelet level in the circulatory system by the methanol extract of P. longifolia also means that it has anticoagulant property.

The relative organ weight is fundamental to diagnose whether the organ was exposed to the injury or not. The heart, liver, kidneys, spleen and lungs are the primary organs affected by metabolic reactions caused by toxicants. The liver is the major site of foreign compounds metabolism in the body (Dybing et al., 2002). The changes observed in the relative weight of the organs such as liver, heart, spleen, kidneys, adrenal glands and testes did not show a dose-dependence effect, it can only represent a normal variation and not as a sign of toxicity (Avancini et al., 2007).

In conclusion, the methanol extract of P. longifolia was well tolerated, lack of mortality and neither produced overt signs of clinical toxicity (diarrhea, loss of hair, behavioral changes, impairments in feed intake and body weight gain), nor any signs of hepato-, nephro-, or hemato-toxicity, also well supported by biochemical data. This acute toxicity study suggests that the methanol extract of P. longifolia is safe up to the dose of 3240 mg/kg b.w. especially when consumed by oral route. Further studies in repeated doses (sub-acute and chronic) must be performed to prove its safety.

Acknowledgement

The authors are thankful to Prof. S.P. Singh, Head, Department of Biosciences, Saurashtra University, Rajkot, Gujarat, India for providing excellent research facilities. The authors Dr. R. D. and Mr. M. K. are thankful to University Grants Commission, New Delhi, India for providing financial support as Junior Research Fellowship.

Declaration of interest

The authors declared no conflict of interest.