Assessment of Neurobehavioral Toxicity of Dendrophthoe falcata (L.f) Ettingsh in Rats by Functional Observational Battery after a Subacute Exposure
SP Pattanayak, P Mitra Mazumder
Division of Pharmacology, Department of Pharmaceutical Sciences, Birla Institute of Technology, Mesra, Ranchi – 835 215, India
|Date of Submission||05-Sep-2008|
|Date of Decision||29-Jan-2009|
|Date of Acceptance||29-Jan-2009|
|Date of Web Publication||30-Dec-2009|
S P Pattanayak
Division of Pharmacology, Department of Pharmaceutical Sciences, Birla Institute of Technology, Mesra, Ranchi – 835 215
Source of Support: None, Conflict of Interest: None
| Abstract|| |
The hemi-parasitic plant Dendrophthoe falcata (L.f) Ettingsh (Loranthaceae) of the order Santalales, is used ethnomedicinally for treating ulcers, asthma, impotence, paralysis, skin diseases, and wounds. The aerial parts are also used in menstrual troubles, psychic disorders, pulmonary tuberculosis, consumption and mania by the tribal of India. In this context, the plant requires the validation of any potential toxicity before therapeutic promotion. The aim of the present study was to evaluate the neurobehavioral toxicity of the hydroalcoholic extract from D. fatcata growing on the host plant Azadirachta indica, after subacute exposure. The LD50 was assessed in female wistar rats and was found to be 4550mg/kg by oral route. The plant extract was administered in three different doses i.e. 250mg/kg, 475mg/kg and 950mg/kg body weight/day for a period of four weeks. At the end of the exposure, behavioural and functional parameters were assessed in a functional observational battery (FOB) and motor activity was measured in an open field. A decrease in the arousal level was observed in experimental groups. Also, the total number of urine spots increased in a dose dependent manner for extract treated groups. Our results suggest that hydroalcoholic extracts from aerial parts of D. falcata should be relatively free from any serious neurobehavioral toxicity and safe to use.
Keywords: Dendrophthoe falcata (L.f) Ettingsh, Hydroalcoholic extracts, Sub-acute exposure, Neurobehavioral toxicity, FOB
|How to cite this article:|
Pattanayak S P, Mazumder P M. Assessment of Neurobehavioral Toxicity of Dendrophthoe falcata (L.f) Ettingsh in Rats by Functional Observational Battery after a Subacute Exposure. Phcog Mag 2009;5:98-105
|How to cite this URL:|
Pattanayak S P, Mazumder P M. Assessment of Neurobehavioral Toxicity of Dendrophthoe falcata (L.f) Ettingsh in Rats by Functional Observational Battery after a Subacute Exposure. Phcog Mag [serial online] 2009 [cited 2022 Jan 24];5:98-105. Available from: http://www.phcog.com/text.asp?2009/5/18/98/57965
| Introduction|| |
Dendrophthoe falcata (L. f) Ettingsh (Loranthaceae), commonly known as 'Banda' (Hindi) is a evergreen shrub with bark smooth grey, leaves opposite unequal, thick 1.6 - 25.4 cm long, flowers single, orange-red or scarlet softly pubescent, berries soft ovoidoblong,1.3cm diameter and indigenous to India, Sritanka, Thailand, Indo-china, Australia  . It is a large bushy parasitic plant that grows on a variety of host plants in deciduous forests and the entire plant is medicinally important  . The aerial parts are used in wounds, menstrual troubles, asthma, psychic disorders, pulmonary tuberculosis, consumption and mania by the tribal of India ,,, . Leaf paste is used in skin diseases  . Its paste is applied on boils, setting dislocated bones and extracting pus  . The plant has been scientifically proved to have antilithiatic, diuretic, cytotoxic and immunomodulatory activities , . In previous phytochemical studies, D. falcata have been reported to contain several cardiac glycosides, flavonoids, and pentacyclic triterpenes , .
In order to continue assessing the potential therapeutic use, it is necessary to investigate their safety through toxicity studies. In the present work, we evaluated the sub-acute neurobehavioral toxicity of hydroalcoholic extract from aerial parts of D. falcata growing on the host plant Azadirachta indica, in rats by means of a functional observational battery (FOB)  and by assessing the motor activity in an open field  . Functional observational battery evaluations in animals are similar to clinical neurologic examinations in humans in that they rate the presence and severity of behavioural and neurologic dysfunction. FOB evaluations in screening typically assess several neurobiologic domains including neuromuscular (weakness, inco-ordination, abnormal movements, gait, motor seizures, myoclonia, rigidity and tremors), sensory (auditory, visual, and somatosensory), and autonomic (pupil response and salivation) functions.
| Materials and Method|| |
Fresh aerial parts of D. falcata were collected in December 2007 from the thick forest areas of Similipal biosphere reserve, Mayurbhanj district of Orissa, India. Dendrophthoe fatcata (L.f) Ettingsh (Loranthaceae) was authenticated by Dr. N.K. Dhal, Department of Natural products, Regional Research Laboratory (RRL), Bhubaneswar, India. Two sets of herbarium voucher specimens were mounted and one set was deposited at RRL, Bhubaneswar vide access no 9996 and one set has been preserved in our laboratory for future reference.
Preparation of extracts
The aerial parts were air-dried, pulverized to a coarse powder in a mechanical grinder, passed through a 40 mesh sieve and extracted in a soxhlet extractor with ethanol-water (8:2). The extract was decanted, filtered with Whatman No. 1 filter paper and concentrated at reduced pressure below 40 ° C through rota vapor to obtain dry extract (20.6% w/w). Dendrophthoe falcata hydroalcoholic extract (DFHE) was kept at 4° C.
Female Wistar rats of 8 weeks old were used for subacute exposure. They were maintained under constant temperature conditions (22 ± 1° C) in a 12-h light:12-h dark cycle (Light on at 07:00), provided with standard food and water ad lib. The experiments were conducted in accordance with the institute's ethical committee approval and guidelines Reg no. 621/02/ac/CPCSEA of Birla Institute of Technology, Mesra, India under the proposal approval no. BIT/PH/IAEC/05/2008.
LD 50 determination
From the acute toxicity study data it was found that at the dose level of 3500mg/kg there was no mortality and at 6000mg/kg all the animals were dead. LD 50 determination of DFHE was performed as described by Graphical method  . Different doses of 3.5, 4, 4.5, 5, 5.5 and 6g/kg were administered orally to the animals of six groups, each containing four animals. The toxicological effect was assessed on the basis of mortality after 24h, which was expressed as an LD 50 value. The percentage of mortality was converted to Probits and the values were plotted against log dose. The LD 50 was the dose intersected by Probit 5.
The plant extract was suspended in 0.3% w/v Sodium carboxy methyl cellulose (Na CMC) in distilled water for experimental use. Three groups of six animals received a daily dose of 250mg/kg of body weight (b.w.) (- 0.05 x LD 50 ), 475mg/kg b.w. (- 0.1 x LD50), and 950mg/kg b.w. (- 0.2 x LD 50 ) of DFHE during a 28day period. Another group formed by 6 rats was used as a control to which vehicle (Na CMC) was administered. In each case the product volume administered was 10ml/kg body weight. The parameter measured during the exposure period was body weight in each alternate day. At the end of the exposure, behavioral and functional parameters and motor activity were assessed in all animals.
Functional observational battery
The FOB includes a through description of the animals' appearance, behaviour and functional integrity (US EPA, 1998). This was assessed through observations in the home cage, while animals were moving freely in an open field, and through manipulative tests. Procedural details and scoring criteria for the FOB protocol have been according to McDaniel and Moser, 1993  with some modifications for rats (Appendix 1).
Briefly, measurements were first carried out in the home cage. The observer recorded each animal's posture, activity and patpebral closure. The presence or absence of tremors and convulsions were noted and, if present, described. The presence or absence of spontaneous vocalizations and biting was also noted. The observer then removed the animal, rating the ease of removal and handling. The presence or absence of hind limb flexor resistance and pressure grade was also noted. Palpebral closure and any lacrimation or salivation were rated. Other abnormal clinical signs were also recorded. The animal was next placed in an open field arena having a piece of clean absorbent paper on the surface and allowed to freely explore for 2 min. During that time, the observer ranked the rat's arousal, gait score, activity level and rears as well as any abnormal postures, unusual movements and stereotypy. At the end of the 3 min, the number of fecal boluses and urine pools and presence or absence of diarrhoea on the absorbent paper was recorded. Next, sensorial responses were ranked according to a variety of stimuli (click stimulus using a metal clicker, approach and touch rump with a blunt object, pinch of the tail using metal tweezer, constriction of the pupil to a penlight stimulus and touch of the corner of the eye with a fine cotton thread). Also, several motor reflexes were evaluated (forelimb hopping, proprioceptive positioning, forelimb and hindlimb extensions). Degree of surface and aerial righting were rated next. In landing foot splay, the tarsal joint pad of each hindfoot was marked with ink and the animal was then dropped from a height of 30 cm onto a recording sheet. This procedure was repeated three times. The distance from center-to-center of the ink marks was measured (cm) and the average of the three splay values was used for statistical analysis.
An open field of 77 cm x 55 cm x 7 cm [l x w x h] whose floor was divided into 12 cm x 12 cm squares by black lines was used. The number of squares entered with all four paws, rearing, grooming and fecal boluses were scored each 5 min for 15 min. After each animal was removed, the open field was carefully cleaned with a damp cloth.
Behavioural test measures in FOB were continuous (providing interval data or count data), ranked (ranked based on a defined scale), descriptive or quantal (presence or absence of sign). Count, ranked and interval data were submitted to a one-way ANOVA. Whereas, the difference between groups in each case were analyzed by Dunnet's t-rest. In all cases, resulting probability values < 0.05 were considered significant.
| Results|| |
LD 50 determination
The LD 50 was also determined by the graphical method and was found to be 4550mg/kg [Table 1], [Figure 1].
Functional observational battery
In the subacute exposure of hydroalcoholic extract from the aerial parts of Dendrophthoe falcata revealed no significant differences in body weight of all the animals (data not shown). The data obtained in the FOB are shown in [Table 1] and [Table 2]. DFHS exposure to the rats produced no alterations in the parameters evaluated in the home cage or during the manipulative tests. Also, no abnormal clinical signs were observed in control and experimental groups. However, in the open field arena both experimental groups exhibited a significant decrease in the arousal level (p < 0.05) compared to control groups. The other parameters evaluated in the open field arena were not altered in the animals exposed. Motor activity evaluations in the square open field indicated that the subacute exposure did not modify the number of squares crossed during a total of 15 min on day 28 [Figure 2]. The ANOVA for repeated measures for comparisons in the number of squares crossed in each 5 min period, did not show significant differences in the number of squares. Moreover, the total number of urine spots were significantly increased in DFHE treated group. The effect followed the expected exposure-response relationships with less significant (p < 0.05) effect in the low exposure (250mg/kg) group, more significant (p < 0.01) effect in the high exposure (950mg/kg) group and transitional response with statistically significant findings, in the intermediate (475 mg/kg) group. When we analysed the emotional parameters as the number of grooming and fecal boluses, no measures demonstrated any significant differences between control and all the experimental groups.
| Discussion|| |
The overall objective of this study was to evaluate the behavioral effects of DFHE exposure in rats. We focused our attention on CNS since the plant is used for the treatment of some psychic disorders. It is also widely used for the treatment of other diseases, so the neurobehavioral parameters were observed to see whether the plant is having any inherent toxicity which if present would make it unsuitable for any therapeutic promotion.
Decrease in the arousal level observed in experimental groups in subacute exposure indicates that the plant may have some depressant like activity, which makes it suitable for the treatment of mania , .
Among the behavioral measurement the most consistent finding was more urine spots in the open field arena, which might be indicating its diuretic property  , but there were no effects on general measures of responding, stimulus control, or disinhibition. The summery of the rodent data publicized that the hydroalcoholic extract of D. falcata might have no effect on neural integrity  which was checked through FOB test and motor activity test indicate that it might be free from neurobehavioral dysfunction . [Table 3]
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[Figure 1], [Figure 2]
[Table 1], [Table 2], [Table 3]
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