- Email: [email protected]
CNS activity of the methanol extract of Mallotus peltatus (Geist) Muell Arg. leaf: an ethnomedicine of Onge
Journal of Ethnopharmacology 85 (2003) 99–105
CNS activity of the methanol extract of Mallotus peltatus (Geist) Muell Arg. leaf: an ethnomedicine of Onge Debprasad Chattopadhyay a,∗ , G. Arunachalam a , Subhash C. Mandal b , R. Bhadra c , Asit B. Mandal d a
b
ICMR Virus Unit Calcutta, Infectious Diseases & Beliaghata General Hospital, GB 4, 1st Floor, 57 Dr. Suresh Chandra Banerjee Road, Kolkata 700 010, India Division of Pharmacognosy, Department of Pharmaceutical Technology, Jadavpur University, Kolkata, India c Cellular Biochemistry Laboratory, Indian Institute of Chemical Biology, Kolkata, India d Biotechnology Laboratory, Central Agricultural Research Institute, Port Blair, Andamans, India Received 1 March 2002; received in revised form 27 November 2002; accepted 27 November 2002
Abstract The aim of the present study was to investigate several neuropharmacological effects of the methanol extract and different fractions of Mallotus peltatus (Geist) Muell Arg. var acuminatus (Euphorbiaceae) leaves in Wistar albino rats and Swiss albino mice. General behavior, exploratory behavior, muscle relaxant activity and phenobarbitone sodium-induced sleeping time were studied. The results revealed that the crude extract at 200–300 mg kg−1 p.o. and its fractions A and B at 50 mg kg−1 caused a significant reduction in spontaneous activity (general behavioral profile), remarkable decrease in exploratory behavioral pattern (Y-maze and head dip tests), a reduction in muscle relaxant activity (rotarod, 30◦ inclined screen and traction tests), and also significantly potentiated phenobarbitone sodium-induced sleeping time. The phytochemical study of crude leaf extract revealed the presence of tannin, triterpenoid, flavonoid, sterol, alkaloid and reducing sugar. Further fractionation and purification yielded two major fractions A (ursolic acid) and B (-sitosterol) with some fatty acids as the major compounds. The psychopharmacological activity of the crude leaf extracts appeared to be either due to fraction A (50 mg kg−1 ) or a combination of fractions A and B (50 mg kg−1 ) alongwith some fatty acids present in the n-butanol part of methanol extract of M. peltatus leaf (MEMPL). © 2002 Elsevier Science Ireland Ltd. All rights reserved. Keywords: Mallotus peltatus; Methanol extract; CNS activity; Experimental animals
1. Introduction The Mallotus peltatus (Geist) Muell Arg. var acuminatus (Euphorbiaceae), locally known as Pataque and Obottacke by Onge (a Nigrito tribes of Little Andaman Islands), is a panatropical shrub endemic to the inland forest of Chidiyatappu, Baratang, Jarawa Creek, and Interview Islands of Andamans. The plant is ±5 M shrub with reddish brown alternate leaves, glossy green above and pale below, petiole with pubescent mid-rib, leaves 3–8 in. long, ovate to oblong, acuminate. Ethnobotanical studies indicate that the decoction of M. peltatus leaves and stem bark has been used for centuries for many indications including skin wounds, stomachache (Dagar and Dagar, 1991) and intestinal ailments ∗ Corresponding
author. Tel.: +91-33-353-7425; fax: +91-33-353-7424. E-mail addresses: [email protected], [email protected] (D. Chattopadhyay).
(Bhargava, 1983; Chakraborty and Vasudeva Rao, 1988) among the tribal populations of Bay Islands, India. Alcoholic extract of leaves is reported to have antitrematodic activity (Asolkar et al., 1992; The Wealth of India, 1985; Ambasta, 1992). Currently, the antimicrobial and anti-inflammatory activities of M. peltatus leaf extracts have been demonstrated (Chattopadhyay et al., 2002a). However, there is no report on the psychopharmacological activity of this plant, although the decoctions of M. peltatus leaves are extensively used by the Onge to reduce mental tension and to induce sleep (author’s personal experience with the Onge tribes of Dugong Creek, Little Andaman). Therefore, in the light of their use in folklore medicine as sedative and antidepressant agent (as observed by the author), the present study was undertaken for the first time to investigate the CNS activity of the methanol extract of M. peltatus leaf (MEMPL) in mice and rats, particularly in animal models of depression.
0378-8741/02/$ – see front matter © 2002 Elsevier Science Ireland Ltd. All rights reserved. doi:10.1016/S0378-8741(02)00379-3
100
D. Chattopadhyay et al. / Journal of Ethnopharmacology 85 (2003) 99–105
2. Material and methods
2.4. Drugs
2.1. Plant materials
The following drugs were used: chlorpromazine hydrochloride (Indus Pharmaceuticals Limited, India), diazepam (Lupin Laboratories Ltd., India), phenobarbitone sodium (Rhone-Poulenc India Ltd., India), pethidine (Ranbaxy Laboratories Ltd., India) and propelyne glycol (SRL Laboratories, India).
The leaves of M. peltatus (Geist) Muell Arg. var acuminatus were collected from the rain forests of Middle and South Andamans, India, during April, June and October 1999. The voucher specimens have been identified and deposited at the Herbarium Section (Herbarium no. 9219) of the Botanical Survey of India, Andaman & Nicobar Circle, Port Blair, India. 2.2. Extraction and fractionation Coarsely powdered dry leaves (1 kg) were successively extracted in cold with 95% methanol as solvent for 72 h at room temperature. The whole extract was collected in a 5 l conical flask, filtered and the solvent were evaporated to dryness under reduced pressure in a Eyela Rotary Evaporator (Japan) at 40–45 ◦ C. The w/w yield of the prepared extract was 8.9 ± 0.21% with respect to the dry powder. The preliminary phytochemical group test of leaf extract was done by the standard methods (Trease and Evans, 1983; Plummer, 1985; Wallis, 1985). The concentrated extracts were then partitioned between n-butanol and H2 O, while the aqueous portion was lyophilized to dryness (∼30 g). The phytochemical tests of the whole extract were done by qualitative analysis and confirmed by thin layer chromatography (Pollock and Stevens, 1965). The crude extract was stored in a dessicator, and the weighed amount was dissolved in propylene glycol, as the extract was fully soluble to it, just before the tests. Further fractionation and purification of n-butanol part of the crude extract yielded two major fractions: (A) triterpene and (B) phytosterol, with some minor fatty acids (Chattopadhyay et al., 2002a). 2.3. Animals used Swiss albino mice (20–25 g) and Wistar albino rats (150–180 g) of either sex were used. The animals were housed in groups of 10 per cage (standard metal cages) prior to pharmacological studies with free access to standard diet and water ad libitum for at least 2 weeks on a 12/12 h light/dark cycle (from 08:00 to 20:00 h). All animals were fasted overnight before test, tap water was supplied ad libitum. The ambient temperature was 22 ± 1 ◦ C, except phenobarbitone sodium-induced sleeping time experiments, which were carried out at 30 ± 1 ◦ C. Plant extracts and standard drug were suspended in propylene glycol immediately prior to use and given orally 1 h before the experiments in a dose of 5 ml kg−1 body weight in mice (0.1 ml/20 g) and rats (0.75 ml/150 g). Control animals received the same dose of vehicle under the same conditions. Behavioral observations took place between 08:00 and 15:00 h and each animal was used only once. Injections were normally made intraperitoneally unless otherwise mentioned.
2.5. LD50 in mice An acute toxicity study was done by determining LD50 , calculated from the lethality within 3 days after p.o. administration, of different doses of the crude extract of M. peltatus leaf by Litchfield and Wilcoxon (1949) method. 2.6. General behavioral profiles Evaluation of general behavioral profile was performed by the method of Dixit and Varma (1976). Seventy adult albino male mice were divided into seven groups (n = 10). The first three groups of animals were administered with MEMPL at the doses of 100, 200 and 300 mg kg−1 intraperitoneally. The fourth and fifth groups were administered with 50 mg kg−1 of fractions A and B, respectively. While the last two groups receive either chlorpromazine (5 mg kg−1 ) as drug control or propylene glycol (5 ml kg−1 ) as vehicle control. The animals were under observation for their behavioral changes if any, at 30 min intervals in the first hour and at hourly intervals for the next 4 h for the following parameters (Mukherjee et al., 1996; Murugesan et al., 1999) 2.6.1. Awareness, alertness and spontaneous activity The awareness and alertness was recorded by visual measure of the animal’s response when placed in different positions and its ability to orient itself without bumps or falls (Turner, 1965). The normal behavior at resting position was scored as 0, little activity (+), moderate flexibility (++), strong response (+++) and abnormal restlessness as (++++). The spontaneous activity of the mice was recorded by placing the animal in a bell jar. It usually shows a moderate degree of inquisitive behavior. Less or moderate activity was scored as ++, strong activity as +++. If there is slight or little motion, the score was +, while if the animal sleeps, the score was −. Excessive or very strong inquisitive activity like constant walking or running was scored ++++. A similar test was performed with the same scoring, when the animals are removed from the jar and placed on a table (Turner, 1965; Mukherjee et al., 1996). 2.6.2. Touch response The touch response was recorded by touching the mice with a pencil or forceps at various parts of the body (i.e. on the side of the neck, abdomen and groin).
D. Chattopadhyay et al. / Journal of Ethnopharmacology 85 (2003) 99–105
2.6.3. Pain response The pain response was graded when a small artery clamp was attached to the base of the tail, and response was noted. 2.6.4. Sound response Albino mice normally utter no sound, so that vocalization may indicate a noxious stimulus. 2.7. Analgesic activity Analgesic activity was studied by (i) tail immersion and (ii) tail flick tests. 2.7.1. Tail immersion test Swiss albino mice of either sex were divided into 7 groups of 10 animals each. Propylene glycol (5 ml kg−1 ), MEMPL at the doses of 100, 200, and 300 mg kg−1 , fractions A and B at doses of 50 mg kg−1 and pethidine (5 mg kg−1 ) were administered intraperitonialy. The tail (up to 5 cm) was then dipped into a pot of water maintained at 55 ± 0.5 ◦ C. The time in seconds to withdraw the tail out of water was taken as the reaction time. The reading was taken after 30 min of administration of the test drugs (Ghosh, 1984). 2.7.2. Tail flick test Wistar strain of albino rats of either sex weighing between 150 and 180 g were selected and divided into 7 groups of 10 animals each. The tail of the rat was place on the nichrome wire of an analgesiometer (Techno, Lucknow, India) and the time taken by the animal to withdraw (flick) its tail from the hot wire was taken as the reaction time. The MEMPL in doses of 100, 200, and 300 mg kg−1 , fractions A and B at doses of 50 mg kg−1 and pethidine (5 mg kg−1 ) were injected intraperitonialy. Propylene glycol at 5 ml kg−1 was served as control. Analgesic activity was measured after 30 min of administration of test and standard drugs (Ghosh, 1984).
101
(5 ml kg−1 ), MEMPL (100, 200, and 300 mg kg−1 ), fractions A and B (50 mg kg−1 ), or diazepam (10 mg kg−1 ), respectively. The rats were placed individually in a symmetrical Y-shaped runway (33 cm × 38 cm × 13 cm) for 3 min and the number of times a rat entered in the arm of the maze with all 4 ft (an ‘entry’) were counted (Rushton et al., 1961; Mandal et al., 2001). 2.9.2. Head dip test Seven groups of female albino mice (n = 10) were placed on top of a wooden box with 16 evenly spaced holes, 30 min after injection of the MEMPL (100, 200, and 300 mg kg−1 ), fractions A and B (50 mg kg−1 ), vehicle (5 ml kg−1 propylene glycol) and diazepam (10 mg kg−1 ) respectively. The number of times that each animal dipped its head into the holes was counted for a period of 3 min (Dorr et al., 1971). 2.10. Muscle relaxant activity The effect of extract on muscle relaxant activity was studied by the (a) traction test, (b) rotarod test, and (c) 30◦ inclined screen test. 2.10.1. Traction test The screening of animals was done by placing the forepaws of the male mice in a small twisted wire rigidly supported above a bench top. Normally the mice grasp the wire with the forepaws, and place at least one hind foot on the wire within 5 s when allowed to hang free. The test was conducted on seven groups of animals (n = 10) which were previously screened, 30 min after the injection of either MEMPL (100, 200, and 300 mg kg−1 ), fractions (50 mg kg−1 ), diazepam (10 mg kg−1 ) or propylene glycol (5 ml kg−1 ) as vehicle control. Inability to put up at least one hind foot considered failure in the traction test (Rudzik et al., 1973).
This was performed by (i) Y-maze and (ii) head dip tests.
2.10.2. Rotarod test Fresh mice were placed on a horizontal wooden rod (32 mm diameter) rotating at a speed of 5 rpm. The mice capable of remaining on the top for 3 min or more, in three successive trials were selected for the study. The selected animals were divided into seven groups (n = 10). Groups 1, 2 and 3 were injected intraperitoneally with MEMPL at 100, 200, and 300 mg kg−1 . Groups 4 and 5 were received either propylene glycol (5 ml kg−1 ) or diazepam (10 mg kg−1 ). While the sixth and seventh group received 50 mg kg−1 of fractions A or B, respectively. Each group of the animals was then placed on the rod at an interval of 30, 60, 90, 120, and 150 min. The animals failed more than once to remain on the rotating rod for 3 min were considered as positive result (Dunham and Miya, 1957).
2.9.1. Y-maze test This was performed in groups of 10 albino rats at 30, 60, 90, and 120 min after injection of either propylene glycol
2.10.3. 30◦ Inclined screen test In this test the groups of 10 male mice, in 7 groups, were injected intraperitoneally with either propylene glycol
2.8. Effect of phenobarbitone sodium-induced sleeping time Mice were divided into 6 groups of 10 each. Animals received 40 mg kg−1 (i.p.) phenobarbitone sodium 30 min after the injection of MEMPL at a dose of 100, 200 and 300 mg kg−1 , fractions A and B at 50 mg kg−1 and control vehicle propylene glycol (5 ml kg−1 ). The sleeping time was recorded, and measured as the time interval between the loss and regaining of the righting reflex (Dandiya and Collumbine, 1959; Mandal et al., 2001). 2.9. Exploratory behavior
102
D. Chattopadhyay et al. / Journal of Ethnopharmacology 85 (2003) 99–105
(5 ml kg−1 ) or MEMPL (100, 200, and 300 mg kg−1 ) or 50 mg kg−1 of fractions A and B or diazepam (10 mg kg−1 ), respectively. The animals were left on a 30◦ inclined screen at least for 4 h to observe a paralysant effect sufficient to cause the mouse slide off the screen (Randall, 1960). 2.11. Statistical analysis The data were expressed as the mean ± S.E. Significance was evaluated by Student’s t-test in all the experiments, and Chi-square tests for muscle relaxant activity (Woodson, 1987). A value less than 0.05 was considered significant.
Table 2 Analgesic effect of MEMPL on tail flick and tail immersion tests in mice and rats Treatment
Dose
Tail flick test (reaction time, s)
Tail immersion test (reaction time, s)
Propylene glycol Aspirin MEMPL
5 ml kg−1
2.25 ± 0.14
2.32 ± 0.16
100 mg kg−1 100 mg kg−1 200 mg kg−1 300 mg kg−1 50 mg kg−1 50 mg kg−1
4.20 2.50 3.02 3.73 3.69 3.01
Fraction A Fraction B
± 0.18 ± 0.13 ± 0.06 ± 0.10 ±0.21 ± 0.12
4.48 2.48 3.05 3.65 3.65 3.02
± ± ± ± ± ±
0.12 0.02 0.02 0.16 0.16 0.12
Values are mean ± S.E., n = 10. All the data are significant at P < 0.001 vs. control, Student’s t-test.
significantly increased in treated animals compared with the standard drug pethidine.
3. Results
3.4. Exploratory behavior potentials
3.1. Toxicity study The leaf extract was found to be non-toxic upto doses of 3.2 g kg−1 and did not cause any death of the animals tested. 3.2. Effect on general behavioral profiles The results obtained from different experiments are presented in Table 1. The MEMPL affected spontaneous activity, sound and touches responses at doses above 200 mg kg−1 and produced moderate or slight depression relating to awareness and alertness. However, the standard drug chlorpromazine hydrochloride, caused a significant depression of all these responses compared with the methanolic extract. The interesting finding is the significant response towards the general behavior of animals with fraction A of the extract.
In Y-maze test, the animals treated with MEMPL in doses of 100 mg kg−1 and above showed a marked decrease in exploratory behavior compared with controls (Table 3). In head dip test, there was a significant reduction in the head dip responses occurred in mice treated with MEMPL at doses of 100 mg kg−1 and above as well as with fraction A, compared with the control (Table 4). 3.5. Effect on phenobarbitone sodium-induced sleeping time The MEMPL significantly potentiated the phenobarbitone sodium-induced sleeping time at the dose of 100 mg kg−1 and above with respect to the control drug diazepam. While the fraction A at 50 mg kg−1 dose showed much better results compared with the control group (Table 5).
3.3. Analgesic activity
3.6. Effect on muscle relaxant activity
The result of the analgesic activity of MEMPL and its fractions by tail immersion and tail flick methods is presented in Table 2. In both the tests the reaction time was
In the traction test, the mice treated with MEMPL showed a significant failure in traction at all doses tested. The result obtained from the rotarod test, showed that MEMPL at
Table 1 Effect of the MEMPL on general behavioral profiles in mice and rats Behavior type
Spontaneous activity Alertness Awareness Sound response Touch response Pain response
MEMPL (mg kg−1 ) 100
200
300
+ + + + ++ +
++ +++ +++ ++ +++ +++
+++ +++ ++ ++++ +++ ++++
Fraction A (50 mg kg−1 )
Fraction B (50 mg kg−1 )
Chlorpromazine (5 mg kg–1 )
Propylene glycol (5 ml kg−1 )
++++ +++ +++ +++ +++ +++
++ +++ +++ ++ +++ +++
++++ +++ +++ ++++ ++++ ++++
− − − − − −
MEMPL, methanol extract of M. peltatus leaf; −, no effect; +, slight depression; ++, moderate depression; +++, strong depression; ++++, very strong depression; n = 10.
D. Chattopadhyay et al. / Journal of Ethnopharmacology 85 (2003) 99–105
103
Table 3 Effect of M. peltatus leaf extract on exploratory behavior (Y-maze test) in rats Experiment
Dose
Number of entries after treatment (min) 30
Propylene glycol Diazepam MEMPL
Fraction A Fraction B
5 ml kg−1 10 mg kg−1 100 mg kg−1 200 mg kg−1 300 mg kg−1 50 mg kg−1 50 mg kg−1
9.2 3.0 6.4 5.0 3.5 3.6 5.8
60 ± ± ± ± ± ± ±
0.2 0.1 0.05 0.2 0.05 0.02 0.2
9.3 3.1 6.4 5.0 3.5 3.55 5.78
90 ± ± ± ± ± ± ±
0.2 0.1 0.05 0.2 0.05 0.01 0.1
9.3 3.2 6.6 5.1 3.6 3.4 5.9
120 ± ± ± ± ± ± ±
0.2 0.1 0.05 0.2 0.05 0.03 0.1
9.4 3.3 6.7 5.1 3.6 3.5 5.4
± ± ± ± ± ± ±
0.2 0.1 0.05 0.2 0.05 0.02 0.2
MEMPL, methanol extract of M. peltatus leaf. Values are the number of entries in 3 min (mean ± S.E., n = 10). All the data are significant at P < 0.001 compared with control. Table 4 Effect of MEMPL on exploratory behavior (head dip test) in mice
Table 6 Percentage effect of the MEMPL on muscle relaxant activity in mice
Experiment
Dose (body weight)
Head dip test
Treatment
Propylene glycol Diazepam MEMPL
5 ml kg−1
99 32 71 60 33 32 72
Fraction A Fraction B
10 mg kg−1 100 mg kg−1 200 mg kg−1 300 mg kg−1 50 mg kg−1 50 mg kg−1
± ± ± ± ± ± ±
1.0 2.0∗∗∗ 3.0∗ 2.0∗∗ 1.0∗∗∗ 1.1∗∗∗ 1.0∗∗
MEMPL, methanol extract of M. peltatus leaf. Values are the number of head dips in 3 min (mean ± S.E., n = 10). ∗ P < 0.05 compared with control. ∗∗ P < 0.01 compared with control. ∗∗∗ P < 0.001 compared with control.
100 mg kg−1 and above significantly reduced the motor coordination of the tested animals; while in 30◦ inclined screen test, the extract showed a significant loss of coordination and muscle tone of all the tested animals. This activity appears to be significantly lower with fraction A at 50 mg kg−1 (Table 6). The results of the preliminary phytochemical group test of M. peltatus leaf extract have been presented in Table 7. The phytochemical tests with the MEMPL indicated the presence of tannin, triterpenoid, flavonoid, alkaloid (trace), sterol and reducing sugar. While further fractionation of n-butanol part of crude methanol extract showed the presence of two
Dose
Traction test
Propylene glycol 5 ml kg−1 0 Diazepam 10 mg kg−1 100 MEMPL 100 mg kg−1 60∗ 200 mg kg−1 70∗ 300 mg kg−1 80∗ Fraction A 50 mg kg−1 80∗ Fraction B 50 mg kg−1 60∗
30◦ Inclined screen test
Rotarod test
0 100 50∗ 60∗ 70∗ 70∗ 50∗
0 100 60∗ 70∗ 80∗ 70∗ 60∗
MEMPL, methanol extract of M. peltatus leaf. Values are the percentage animals showing a negative result; n = 10. ∗ P < 0.05 compared with control (Chi-square test).
Table 7 Preliminary phytoconstituents in MEMPL Serial No.
Phytoconstituents
Leaf extract of M. peltatus
1 2 3 4 5 6 7 8 9 10 11
Alkaloids Triterpenoids Steroids Amino Acids Flavonoids Gums Reducing sugars Tannins Saponins Fraction A Fraction B
+ (in traces) + + − + − + + + Ursolic acid -Sitosterol, minor fatty acids
−, Absence; +, presence.
Table 5 Effect of MEMPL on phenobarbitone sodium-induced sleeping time Experiment
Dose
Sleeping time (min)
Propylene glycol MEMPL
5 ml kg−1
64 70 82 119 114 75
Fraction A Fraction B
100 mg kg−1 200 mg kg−1 300 mg kg−1 50 mg kg−1 50 mg kg−1
± ± ± ± ± ±
0 2.0∗ 3.0∗ 3.0∗ 1.1∗ 2.0∗
MEMPL, methanol extract of M. peltatus leaf. Values are mean ± S.E., n = 10. ∗ P < 0.001 compared with control.
major group of compounds namely, fraction A (ursolic acid, triterpene), fraction B (-sitosterol and some fatty acid).
4. Discussion The results indicated that the MEMPL influences general behavioral profiles, as evidenced in the spontaneous activity, touch, sound and pain responses. The MEMPL possess significant analgesic activity compared with the standard drug
104
D. Chattopadhyay et al. / Journal of Ethnopharmacology 85 (2003) 99–105
pethidine in a dose-dependent manner. This activity may be due to its action on central nervous system like pethidine. M. peltatus leaf extract was reported to decrease the normal body temperature and yeast provoked elevation of body temperature significantly in a dose-dependent manner at 100–300 mg kg–1 doses (Chattopadhyay et al., 2002b). The extract significantly potentiated the phenobarbitone sodium-induced sleeping time, possibly through a CNS depressant action (Fastier et al., 1957) or a tranquilizing action (Mandal et al., 2001). The possible CNS activity of the methanolic extract, as well as the major fractions, was further investigated by other common psychopharmacological tests like the rotarod test, 30◦ inclined screen test and traction test. The reduction in exploratory behavior in animals treated with the methanolic crude extract or fractions A, and B of M. peltatus leaf is similar with the action of other CNS depressant agents. A significant lack in motor coordination and muscle relaxant activity was also noted in animals treated with the crude extract as well as with the fraction A. It has been reported that species of Mallotus (M. philippinensis, M. japonicus, M. repandus) contain flavonoids (mallotus A and B), terpenoids (mallotucin and malloripine; diterpenic lactones mallotucin C and D), saponins (rhamnoside crotoxigenin and coroglusagenine), cardinolipids, rottlerine and isorottlerine (The Wealth of India, 1985; Ambasta, 1992). But until recently, there are no reports on the chemistry or pharmacology of M. peltatus var acuminatus. For the first time, we have isolated two major compounds, ursolic acid (fraction A) and -sitosterol alongwith some fatty acids (fraction B). Contemporary scientific research revealed that the ursolic acid isolated from herbal sources have several pharmacological actions, like anticancerous (Kim, 1997), antiulcer, antihyperlipidemic, anti-inflammatory, hepatoprotective, antimicrobial and antiviral (Liu, 1995; Sattar et al., 1995; Zaletova et al., 1987; Chattopadhyay et al., 2001, 2002a). Ursolic acid isolated from several medicinal plants is found to inhibit human leukocyte elastase (Safayhi et al., 1997), 5-lipooxygenase and cyclooxygenase (Najid et al., 1992), concavalin A-induced histamine release (Tsuruga et al., 1991), and enzymes of arachidonate cascade (Diaz et al., 2000). The -sitosterol, on the other hand, reported to have anticancerous, antiulcer, antidiabetic, anti-inflammatory and antipyretic activities (Gupta et al., 1996; Pegel, 1980), and is helpful in controlling rheumatoid arthritis (Pegel, 1980). The present investigation for the first time, confirms that there is a moderate to strong degree of antipsychotic activity in the MEMPL, which appears to be due to the action of either tritepenes (fraction A) alone, or the combination of tritepenes and -sitosterol (fraction B) present in the methanol extract. In conclusion, the vernacular medicinal uses of M. peltatus leaf for mental tension and disturbance as well as to induce sleep is validated by our findings.
Acknowledgements The authors are grateful to the Department of Biotechnology, Government of India, for financial assistance through the project Grant No. BT/PRO 237/OSC 17/005/96. Thanks are due to Dr. Sreekumar, Senior Scientist, and the Officer In-Charge, Botanical Survey of India, Andaman & Nicobar Circle, Port Blair, Andamans for identification of the plant species. We express our gratitude to Dr. S.K. Bhattacharya, Director, National Institute of Cholera & Enteric Diseases, Kolkata and Officer In-Charge, ICMR Virus Unit Calcutta, for his constant encouragement, help and suggestions.
References Ambasta, S.P. (Ed.), 1992. The Useful Plants of India. Publications & Information Directorate (P & ID), CSIR, New Delhi, pp. 31–32. Asolkar, L.V., Kakkar, K.K., Chakre, O.J. (Eds.), 1992. Second Supplement to Glossary of Indian Medicinal Plants with Active Principles, Part 1 (A-K). Publications & Information Directorate, CSIR, New Delhi, pp. 51–52. Bhargava, N., 1983. Ethnobotanical studies of the tribes of Andaman and Nicobar Islands, India. I. Onge. Economic Botany 37, 110–119. Chakraborty, T., Vasudeva Rao, M.K., 1988. Ethnobotanical studies of the Shompen of Great Nicobar Islands. Journal of Economic and Taxonomic Botany 12 (1), 43–54. Chattopadhyay, D., Arunachalam, G., Mandal, A.B., Sur, T.K., Mandal, S.C., Bhattacharya, S.K., 2002a. Antimicrobial and antiinflammatory activity of folklore Mallotus peltatus leaf extract. Journal of Ethnopharmacology 82, 229–237. Chattopadhyay, D., Arunachalam, G., Mandal, A.B., Mandal, S.C., 2002b. Evaluation of antipyretic activity of leaf extract of Mallotus peltatus (Geist) Muell Arg. var acuminatus: a folk medicine. Phytomedicine 9, 727–730. Chattopadhyay, D., Maiti, K., Kundu, A.P., Chakrabarty, M.S., Bhadra, R., Mandal, S.C., Bhattacharya, S.K., 2001. Antimicrobial activity of Alstonia macrophylla: a folklore of Bay Islands. Journal of Ethnopharmacology 77, 49–55. Dagar, H.S., Dagar, J.C., 1991. Plant folk medicines among Nicobarese of Katchal Island, India. Economic Botany 45 (I), 114–119. Dandiya, P.C., Collumbine, H., 1959. Studies on Acorus calamus (III): some pharmacological action of the volatile oil. Journal of Pharmacology and Experimental Therapeutics 125, 353–359. Diaz, A.M., Abad, M.J., Fernandez, L., Recuero, C., Villaescusa, L., Silvan, A.M., Bermejo, P., 2000. In vitro anti-inflammatory activity of iridoids and triterpenoid compounds isolated from Phillyrea latifolia L. Biological & Pharmaceutical Bulletin 23 (11), 1307–1313. Dixit, V.K., Varma, K.C., 1976. Effects of essential oil of leaves of Blumea lacera DC on central nervous system. Indian Journal of Pharmacology 18, 7–11. Dorr, M., Stienberg, H., Tomkiewiez, M., Joyee, D., Porosolt, R.D., Summerfield, A., 1971. Persistence of dose related behaviour in mice. Nature 231, 121–123. Dunham, N.W., Miya, T.S., 1957. A note on simple apparatus for detecting neurological deficit in rats and mice. Journal of American Pharmacology 46, 208–209. Fastier, F.N., Spenden, R.N., Hendrieka, W., 1957. Prolongation of chloral hydrate sleeping time by 5-HT and by certain other drugs. British Journal of Pharmacology 12, 251–259. Ghosh, M.N. (Eds.), 1984. Fundamental of Experimental Pharmacology, 2nd ed. Scientific Book Agency, Calcutta, p. 153.
D. Chattopadhyay et al. / Journal of Ethnopharmacology 85 (2003) 99–105 Gupta, M.B., Nath, R., Srivastava, N., Shanker, K., Kishor, K., Bhargava, K.P., 1996. Anti-inflammatory and antipyretic activities of -sitosterol. International Journal of Immunopharmacology 18 (12), 693–700. Kim, K.-W., 1997. Anticancer activities of plant triterpenoids, ursolic acid and oleanoid acid. Journal of Korean Association and Cancer Prevention 2, 38–44. Litchfield, J.T., Wilcoxon, F., 1949. A simplified method of evaluating dose–effect experiments. Journal of Pharmacology and Experimental Therapeutics 96, 99–133. Liu, J., 1995. Pharmacology of oleanolic and ursolic acid. Journal of Ethnopharmacology 49, 57–68. Mandal, S.C., Dhara, A.K., Maiti, B.C., 2001. Studies of psychopharmacological activity of Andrographis paniculata extract. Phytotherapy Research 15, 253–256. Mukherjee, P.K., Saha, K., Balasubramanium, R., Pal, M., Saha, B.P., 1996. Studies on psychopharmacological effects of Nelumbo nucifera Gaertn. Rhizome extract. Journal of Ethnopharmacology 54, 63–67. Murugesan, T., Ghosh, L., Das, J., Pal, M., Saha, B.P., 1999. CNS activity of Jussiaea suffruticosa Linn. extract in rats and mice. Pharmacy and Pharmacology Communication 5, 663–666. Najid, A., Simon, A., Cook, J., Chable-Rabinobitch, H., Delage, C., Chulia, A., Riguad, M., 1992. Characterization of ursolic acid as a lipoxygenase and cyclooxygenase inhibitor using macrophages, platelets and differentiated HL 60 leukemic cells. FEBS Letter 299 (3), 213–217. Pegel, K.H., 1980. The importance of sitosterol and sitosterolin in human and animal nutrition. Planta Medica 39, 157–163. Plummer, D.I. (Ed.), 1985. An Introduction to Practical Biochemistry, 2nd ed. Tata Macgrow-Hill Publishing Co. Ltd., New Delhi, pp. 136, 143. Pollock, J.R.A., Stevens, R. (Eds.), 1965. Dictionary of Organic Compounds, vol. 5, 4th ed. Eyre & Spottiswoode Publishers Ltd., London, pp. 127–217. Randall, P., 1960. Studies of the neurological effects using simple experimental model. Journal of Pharmacology and Experimental Therapeutics 135, 543–545.
105
Rudzik, A.D., Hester, J.B., Tang, A.H., Staw, R.N., Friis, W. (Eds.), 1973. The Benzodiazepines. Raven Press, New York, pp. 285– 297. Rushton, R., Steinberg, H., Tinson, C., 1961. Modification of the effects of an amphetamine barbiturate mixture by the past experience of rats (Y-shaped runway). Nature 192, 533–535. Safayhi, H., Rall, B., Sailer, E., Ammon, H., 1997. Inhibition of boswellic acids of human leucocyte elastase. Journal of Pharmacology and Experimental Therapeutics 281 (10), 460–463. Sattar, A., Bankova, V., Kujumgiev, A., Galabov, A., Ignatova, A., Todorova, C., Papov, S., 1995. Chemical composition and biological activity of leaf exudates from some Lamiaceae plants. Pharmazie 50, 62– 68. Trease, G.E., Evans, W.C. (Eds.), 1983. Pharmacognosy, 12th ed. ELBS Publication, Baillier Tindall, East Bourne, pp. 344, 539. Tsuruga, T., Chun, Y., Ebizuka, Y., Sankawa, U., 1991. Biologically active constituents of Melaleuca leucadendron; inhibitors of induced histamine release from rat mast cells. Chemical and Pharmacological Bulletin 39 (12), 3276–3278. Turner, R.A. (Ed.), 1965. Screening Methods in Pharmacology. Academic Press, New York, pp. 26–35. Raw Materials, 1985. Publications & Information Directorate, CSIR, New Delhi, vols. I–XI, 1948–1976; revised and enlarged, vol. 1A, pp. 199–200. Wallis, T.E. (Ed.), 1985. Textbook of Pharmacognosy, 5th ed. CBS Publishers & Distributors, Delhi, pp. 15–20, 561–565. Woodson, R.F. (Ed.), 1987. Statistical Method for the Analysis of Bio-Medical Data, Probability and Mathematical Statistics. Wiley, Chichester, pp. 313–316. Zaletova, N., Shavlinskii, A., Tolkacchaev, O., Vichkanova, S., Fateeva, T., Krutikova, N., Yartseva, I., Klyuev, N., 1987. Preparation of some derivatives of ursolic acid and their antimicrobial activity. Chemical Abstracts 106, 18867e.
CNS activity of the methanol extract of Mallotus peltatus (Geist) Muell Arg. leaf: an ethnomedicine of Onge Debprasad Chattopadhyay a,∗ , G. Arunachalam a , Subhash C. Mandal b , R. Bhadra c , Asit B. Mandal d a
b
ICMR Virus Unit Calcutta, Infectious Diseases & Beliaghata General Hospital, GB 4, 1st Floor, 57 Dr. Suresh Chandra Banerjee Road, Kolkata 700 010, India Division of Pharmacognosy, Department of Pharmaceutical Technology, Jadavpur University, Kolkata, India c Cellular Biochemistry Laboratory, Indian Institute of Chemical Biology, Kolkata, India d Biotechnology Laboratory, Central Agricultural Research Institute, Port Blair, Andamans, India Received 1 March 2002; received in revised form 27 November 2002; accepted 27 November 2002
Abstract The aim of the present study was to investigate several neuropharmacological effects of the methanol extract and different fractions of Mallotus peltatus (Geist) Muell Arg. var acuminatus (Euphorbiaceae) leaves in Wistar albino rats and Swiss albino mice. General behavior, exploratory behavior, muscle relaxant activity and phenobarbitone sodium-induced sleeping time were studied. The results revealed that the crude extract at 200–300 mg kg−1 p.o. and its fractions A and B at 50 mg kg−1 caused a significant reduction in spontaneous activity (general behavioral profile), remarkable decrease in exploratory behavioral pattern (Y-maze and head dip tests), a reduction in muscle relaxant activity (rotarod, 30◦ inclined screen and traction tests), and also significantly potentiated phenobarbitone sodium-induced sleeping time. The phytochemical study of crude leaf extract revealed the presence of tannin, triterpenoid, flavonoid, sterol, alkaloid and reducing sugar. Further fractionation and purification yielded two major fractions A (ursolic acid) and B (-sitosterol) with some fatty acids as the major compounds. The psychopharmacological activity of the crude leaf extracts appeared to be either due to fraction A (50 mg kg−1 ) or a combination of fractions A and B (50 mg kg−1 ) alongwith some fatty acids present in the n-butanol part of methanol extract of M. peltatus leaf (MEMPL). © 2002 Elsevier Science Ireland Ltd. All rights reserved. Keywords: Mallotus peltatus; Methanol extract; CNS activity; Experimental animals
1. Introduction The Mallotus peltatus (Geist) Muell Arg. var acuminatus (Euphorbiaceae), locally known as Pataque and Obottacke by Onge (a Nigrito tribes of Little Andaman Islands), is a panatropical shrub endemic to the inland forest of Chidiyatappu, Baratang, Jarawa Creek, and Interview Islands of Andamans. The plant is ±5 M shrub with reddish brown alternate leaves, glossy green above and pale below, petiole with pubescent mid-rib, leaves 3–8 in. long, ovate to oblong, acuminate. Ethnobotanical studies indicate that the decoction of M. peltatus leaves and stem bark has been used for centuries for many indications including skin wounds, stomachache (Dagar and Dagar, 1991) and intestinal ailments ∗ Corresponding
author. Tel.: +91-33-353-7425; fax: +91-33-353-7424. E-mail addresses: [email protected], [email protected] (D. Chattopadhyay).
(Bhargava, 1983; Chakraborty and Vasudeva Rao, 1988) among the tribal populations of Bay Islands, India. Alcoholic extract of leaves is reported to have antitrematodic activity (Asolkar et al., 1992; The Wealth of India, 1985; Ambasta, 1992). Currently, the antimicrobial and anti-inflammatory activities of M. peltatus leaf extracts have been demonstrated (Chattopadhyay et al., 2002a). However, there is no report on the psychopharmacological activity of this plant, although the decoctions of M. peltatus leaves are extensively used by the Onge to reduce mental tension and to induce sleep (author’s personal experience with the Onge tribes of Dugong Creek, Little Andaman). Therefore, in the light of their use in folklore medicine as sedative and antidepressant agent (as observed by the author), the present study was undertaken for the first time to investigate the CNS activity of the methanol extract of M. peltatus leaf (MEMPL) in mice and rats, particularly in animal models of depression.
0378-8741/02/$ – see front matter © 2002 Elsevier Science Ireland Ltd. All rights reserved. doi:10.1016/S0378-8741(02)00379-3
100
D. Chattopadhyay et al. / Journal of Ethnopharmacology 85 (2003) 99–105
2. Material and methods
2.4. Drugs
2.1. Plant materials
The following drugs were used: chlorpromazine hydrochloride (Indus Pharmaceuticals Limited, India), diazepam (Lupin Laboratories Ltd., India), phenobarbitone sodium (Rhone-Poulenc India Ltd., India), pethidine (Ranbaxy Laboratories Ltd., India) and propelyne glycol (SRL Laboratories, India).
The leaves of M. peltatus (Geist) Muell Arg. var acuminatus were collected from the rain forests of Middle and South Andamans, India, during April, June and October 1999. The voucher specimens have been identified and deposited at the Herbarium Section (Herbarium no. 9219) of the Botanical Survey of India, Andaman & Nicobar Circle, Port Blair, India. 2.2. Extraction and fractionation Coarsely powdered dry leaves (1 kg) were successively extracted in cold with 95% methanol as solvent for 72 h at room temperature. The whole extract was collected in a 5 l conical flask, filtered and the solvent were evaporated to dryness under reduced pressure in a Eyela Rotary Evaporator (Japan) at 40–45 ◦ C. The w/w yield of the prepared extract was 8.9 ± 0.21% with respect to the dry powder. The preliminary phytochemical group test of leaf extract was done by the standard methods (Trease and Evans, 1983; Plummer, 1985; Wallis, 1985). The concentrated extracts were then partitioned between n-butanol and H2 O, while the aqueous portion was lyophilized to dryness (∼30 g). The phytochemical tests of the whole extract were done by qualitative analysis and confirmed by thin layer chromatography (Pollock and Stevens, 1965). The crude extract was stored in a dessicator, and the weighed amount was dissolved in propylene glycol, as the extract was fully soluble to it, just before the tests. Further fractionation and purification of n-butanol part of the crude extract yielded two major fractions: (A) triterpene and (B) phytosterol, with some minor fatty acids (Chattopadhyay et al., 2002a). 2.3. Animals used Swiss albino mice (20–25 g) and Wistar albino rats (150–180 g) of either sex were used. The animals were housed in groups of 10 per cage (standard metal cages) prior to pharmacological studies with free access to standard diet and water ad libitum for at least 2 weeks on a 12/12 h light/dark cycle (from 08:00 to 20:00 h). All animals were fasted overnight before test, tap water was supplied ad libitum. The ambient temperature was 22 ± 1 ◦ C, except phenobarbitone sodium-induced sleeping time experiments, which were carried out at 30 ± 1 ◦ C. Plant extracts and standard drug were suspended in propylene glycol immediately prior to use and given orally 1 h before the experiments in a dose of 5 ml kg−1 body weight in mice (0.1 ml/20 g) and rats (0.75 ml/150 g). Control animals received the same dose of vehicle under the same conditions. Behavioral observations took place between 08:00 and 15:00 h and each animal was used only once. Injections were normally made intraperitoneally unless otherwise mentioned.
2.5. LD50 in mice An acute toxicity study was done by determining LD50 , calculated from the lethality within 3 days after p.o. administration, of different doses of the crude extract of M. peltatus leaf by Litchfield and Wilcoxon (1949) method. 2.6. General behavioral profiles Evaluation of general behavioral profile was performed by the method of Dixit and Varma (1976). Seventy adult albino male mice were divided into seven groups (n = 10). The first three groups of animals were administered with MEMPL at the doses of 100, 200 and 300 mg kg−1 intraperitoneally. The fourth and fifth groups were administered with 50 mg kg−1 of fractions A and B, respectively. While the last two groups receive either chlorpromazine (5 mg kg−1 ) as drug control or propylene glycol (5 ml kg−1 ) as vehicle control. The animals were under observation for their behavioral changes if any, at 30 min intervals in the first hour and at hourly intervals for the next 4 h for the following parameters (Mukherjee et al., 1996; Murugesan et al., 1999) 2.6.1. Awareness, alertness and spontaneous activity The awareness and alertness was recorded by visual measure of the animal’s response when placed in different positions and its ability to orient itself without bumps or falls (Turner, 1965). The normal behavior at resting position was scored as 0, little activity (+), moderate flexibility (++), strong response (+++) and abnormal restlessness as (++++). The spontaneous activity of the mice was recorded by placing the animal in a bell jar. It usually shows a moderate degree of inquisitive behavior. Less or moderate activity was scored as ++, strong activity as +++. If there is slight or little motion, the score was +, while if the animal sleeps, the score was −. Excessive or very strong inquisitive activity like constant walking or running was scored ++++. A similar test was performed with the same scoring, when the animals are removed from the jar and placed on a table (Turner, 1965; Mukherjee et al., 1996). 2.6.2. Touch response The touch response was recorded by touching the mice with a pencil or forceps at various parts of the body (i.e. on the side of the neck, abdomen and groin).
D. Chattopadhyay et al. / Journal of Ethnopharmacology 85 (2003) 99–105
2.6.3. Pain response The pain response was graded when a small artery clamp was attached to the base of the tail, and response was noted. 2.6.4. Sound response Albino mice normally utter no sound, so that vocalization may indicate a noxious stimulus. 2.7. Analgesic activity Analgesic activity was studied by (i) tail immersion and (ii) tail flick tests. 2.7.1. Tail immersion test Swiss albino mice of either sex were divided into 7 groups of 10 animals each. Propylene glycol (5 ml kg−1 ), MEMPL at the doses of 100, 200, and 300 mg kg−1 , fractions A and B at doses of 50 mg kg−1 and pethidine (5 mg kg−1 ) were administered intraperitonialy. The tail (up to 5 cm) was then dipped into a pot of water maintained at 55 ± 0.5 ◦ C. The time in seconds to withdraw the tail out of water was taken as the reaction time. The reading was taken after 30 min of administration of the test drugs (Ghosh, 1984). 2.7.2. Tail flick test Wistar strain of albino rats of either sex weighing between 150 and 180 g were selected and divided into 7 groups of 10 animals each. The tail of the rat was place on the nichrome wire of an analgesiometer (Techno, Lucknow, India) and the time taken by the animal to withdraw (flick) its tail from the hot wire was taken as the reaction time. The MEMPL in doses of 100, 200, and 300 mg kg−1 , fractions A and B at doses of 50 mg kg−1 and pethidine (5 mg kg−1 ) were injected intraperitonialy. Propylene glycol at 5 ml kg−1 was served as control. Analgesic activity was measured after 30 min of administration of test and standard drugs (Ghosh, 1984).
101
(5 ml kg−1 ), MEMPL (100, 200, and 300 mg kg−1 ), fractions A and B (50 mg kg−1 ), or diazepam (10 mg kg−1 ), respectively. The rats were placed individually in a symmetrical Y-shaped runway (33 cm × 38 cm × 13 cm) for 3 min and the number of times a rat entered in the arm of the maze with all 4 ft (an ‘entry’) were counted (Rushton et al., 1961; Mandal et al., 2001). 2.9.2. Head dip test Seven groups of female albino mice (n = 10) were placed on top of a wooden box with 16 evenly spaced holes, 30 min after injection of the MEMPL (100, 200, and 300 mg kg−1 ), fractions A and B (50 mg kg−1 ), vehicle (5 ml kg−1 propylene glycol) and diazepam (10 mg kg−1 ) respectively. The number of times that each animal dipped its head into the holes was counted for a period of 3 min (Dorr et al., 1971). 2.10. Muscle relaxant activity The effect of extract on muscle relaxant activity was studied by the (a) traction test, (b) rotarod test, and (c) 30◦ inclined screen test. 2.10.1. Traction test The screening of animals was done by placing the forepaws of the male mice in a small twisted wire rigidly supported above a bench top. Normally the mice grasp the wire with the forepaws, and place at least one hind foot on the wire within 5 s when allowed to hang free. The test was conducted on seven groups of animals (n = 10) which were previously screened, 30 min after the injection of either MEMPL (100, 200, and 300 mg kg−1 ), fractions (50 mg kg−1 ), diazepam (10 mg kg−1 ) or propylene glycol (5 ml kg−1 ) as vehicle control. Inability to put up at least one hind foot considered failure in the traction test (Rudzik et al., 1973).
This was performed by (i) Y-maze and (ii) head dip tests.
2.10.2. Rotarod test Fresh mice were placed on a horizontal wooden rod (32 mm diameter) rotating at a speed of 5 rpm. The mice capable of remaining on the top for 3 min or more, in three successive trials were selected for the study. The selected animals were divided into seven groups (n = 10). Groups 1, 2 and 3 were injected intraperitoneally with MEMPL at 100, 200, and 300 mg kg−1 . Groups 4 and 5 were received either propylene glycol (5 ml kg−1 ) or diazepam (10 mg kg−1 ). While the sixth and seventh group received 50 mg kg−1 of fractions A or B, respectively. Each group of the animals was then placed on the rod at an interval of 30, 60, 90, 120, and 150 min. The animals failed more than once to remain on the rotating rod for 3 min were considered as positive result (Dunham and Miya, 1957).
2.9.1. Y-maze test This was performed in groups of 10 albino rats at 30, 60, 90, and 120 min after injection of either propylene glycol
2.10.3. 30◦ Inclined screen test In this test the groups of 10 male mice, in 7 groups, were injected intraperitoneally with either propylene glycol
2.8. Effect of phenobarbitone sodium-induced sleeping time Mice were divided into 6 groups of 10 each. Animals received 40 mg kg−1 (i.p.) phenobarbitone sodium 30 min after the injection of MEMPL at a dose of 100, 200 and 300 mg kg−1 , fractions A and B at 50 mg kg−1 and control vehicle propylene glycol (5 ml kg−1 ). The sleeping time was recorded, and measured as the time interval between the loss and regaining of the righting reflex (Dandiya and Collumbine, 1959; Mandal et al., 2001). 2.9. Exploratory behavior
102
D. Chattopadhyay et al. / Journal of Ethnopharmacology 85 (2003) 99–105
(5 ml kg−1 ) or MEMPL (100, 200, and 300 mg kg−1 ) or 50 mg kg−1 of fractions A and B or diazepam (10 mg kg−1 ), respectively. The animals were left on a 30◦ inclined screen at least for 4 h to observe a paralysant effect sufficient to cause the mouse slide off the screen (Randall, 1960). 2.11. Statistical analysis The data were expressed as the mean ± S.E. Significance was evaluated by Student’s t-test in all the experiments, and Chi-square tests for muscle relaxant activity (Woodson, 1987). A value less than 0.05 was considered significant.
Table 2 Analgesic effect of MEMPL on tail flick and tail immersion tests in mice and rats Treatment
Dose
Tail flick test (reaction time, s)
Tail immersion test (reaction time, s)
Propylene glycol Aspirin MEMPL
5 ml kg−1
2.25 ± 0.14
2.32 ± 0.16
100 mg kg−1 100 mg kg−1 200 mg kg−1 300 mg kg−1 50 mg kg−1 50 mg kg−1
4.20 2.50 3.02 3.73 3.69 3.01
Fraction A Fraction B
± 0.18 ± 0.13 ± 0.06 ± 0.10 ±0.21 ± 0.12
4.48 2.48 3.05 3.65 3.65 3.02
± ± ± ± ± ±
0.12 0.02 0.02 0.16 0.16 0.12
Values are mean ± S.E., n = 10. All the data are significant at P < 0.001 vs. control, Student’s t-test.
significantly increased in treated animals compared with the standard drug pethidine.
3. Results
3.4. Exploratory behavior potentials
3.1. Toxicity study The leaf extract was found to be non-toxic upto doses of 3.2 g kg−1 and did not cause any death of the animals tested. 3.2. Effect on general behavioral profiles The results obtained from different experiments are presented in Table 1. The MEMPL affected spontaneous activity, sound and touches responses at doses above 200 mg kg−1 and produced moderate or slight depression relating to awareness and alertness. However, the standard drug chlorpromazine hydrochloride, caused a significant depression of all these responses compared with the methanolic extract. The interesting finding is the significant response towards the general behavior of animals with fraction A of the extract.
In Y-maze test, the animals treated with MEMPL in doses of 100 mg kg−1 and above showed a marked decrease in exploratory behavior compared with controls (Table 3). In head dip test, there was a significant reduction in the head dip responses occurred in mice treated with MEMPL at doses of 100 mg kg−1 and above as well as with fraction A, compared with the control (Table 4). 3.5. Effect on phenobarbitone sodium-induced sleeping time The MEMPL significantly potentiated the phenobarbitone sodium-induced sleeping time at the dose of 100 mg kg−1 and above with respect to the control drug diazepam. While the fraction A at 50 mg kg−1 dose showed much better results compared with the control group (Table 5).
3.3. Analgesic activity
3.6. Effect on muscle relaxant activity
The result of the analgesic activity of MEMPL and its fractions by tail immersion and tail flick methods is presented in Table 2. In both the tests the reaction time was
In the traction test, the mice treated with MEMPL showed a significant failure in traction at all doses tested. The result obtained from the rotarod test, showed that MEMPL at
Table 1 Effect of the MEMPL on general behavioral profiles in mice and rats Behavior type
Spontaneous activity Alertness Awareness Sound response Touch response Pain response
MEMPL (mg kg−1 ) 100
200
300
+ + + + ++ +
++ +++ +++ ++ +++ +++
+++ +++ ++ ++++ +++ ++++
Fraction A (50 mg kg−1 )
Fraction B (50 mg kg−1 )
Chlorpromazine (5 mg kg–1 )
Propylene glycol (5 ml kg−1 )
++++ +++ +++ +++ +++ +++
++ +++ +++ ++ +++ +++
++++ +++ +++ ++++ ++++ ++++
− − − − − −
MEMPL, methanol extract of M. peltatus leaf; −, no effect; +, slight depression; ++, moderate depression; +++, strong depression; ++++, very strong depression; n = 10.
D. Chattopadhyay et al. / Journal of Ethnopharmacology 85 (2003) 99–105
103
Table 3 Effect of M. peltatus leaf extract on exploratory behavior (Y-maze test) in rats Experiment
Dose
Number of entries after treatment (min) 30
Propylene glycol Diazepam MEMPL
Fraction A Fraction B
5 ml kg−1 10 mg kg−1 100 mg kg−1 200 mg kg−1 300 mg kg−1 50 mg kg−1 50 mg kg−1
9.2 3.0 6.4 5.0 3.5 3.6 5.8
60 ± ± ± ± ± ± ±
0.2 0.1 0.05 0.2 0.05 0.02 0.2
9.3 3.1 6.4 5.0 3.5 3.55 5.78
90 ± ± ± ± ± ± ±
0.2 0.1 0.05 0.2 0.05 0.01 0.1
9.3 3.2 6.6 5.1 3.6 3.4 5.9
120 ± ± ± ± ± ± ±
0.2 0.1 0.05 0.2 0.05 0.03 0.1
9.4 3.3 6.7 5.1 3.6 3.5 5.4
± ± ± ± ± ± ±
0.2 0.1 0.05 0.2 0.05 0.02 0.2
MEMPL, methanol extract of M. peltatus leaf. Values are the number of entries in 3 min (mean ± S.E., n = 10). All the data are significant at P < 0.001 compared with control. Table 4 Effect of MEMPL on exploratory behavior (head dip test) in mice
Table 6 Percentage effect of the MEMPL on muscle relaxant activity in mice
Experiment
Dose (body weight)
Head dip test
Treatment
Propylene glycol Diazepam MEMPL
5 ml kg−1
99 32 71 60 33 32 72
Fraction A Fraction B
10 mg kg−1 100 mg kg−1 200 mg kg−1 300 mg kg−1 50 mg kg−1 50 mg kg−1
± ± ± ± ± ± ±
1.0 2.0∗∗∗ 3.0∗ 2.0∗∗ 1.0∗∗∗ 1.1∗∗∗ 1.0∗∗
MEMPL, methanol extract of M. peltatus leaf. Values are the number of head dips in 3 min (mean ± S.E., n = 10). ∗ P < 0.05 compared with control. ∗∗ P < 0.01 compared with control. ∗∗∗ P < 0.001 compared with control.
100 mg kg−1 and above significantly reduced the motor coordination of the tested animals; while in 30◦ inclined screen test, the extract showed a significant loss of coordination and muscle tone of all the tested animals. This activity appears to be significantly lower with fraction A at 50 mg kg−1 (Table 6). The results of the preliminary phytochemical group test of M. peltatus leaf extract have been presented in Table 7. The phytochemical tests with the MEMPL indicated the presence of tannin, triterpenoid, flavonoid, alkaloid (trace), sterol and reducing sugar. While further fractionation of n-butanol part of crude methanol extract showed the presence of two
Dose
Traction test
Propylene glycol 5 ml kg−1 0 Diazepam 10 mg kg−1 100 MEMPL 100 mg kg−1 60∗ 200 mg kg−1 70∗ 300 mg kg−1 80∗ Fraction A 50 mg kg−1 80∗ Fraction B 50 mg kg−1 60∗
30◦ Inclined screen test
Rotarod test
0 100 50∗ 60∗ 70∗ 70∗ 50∗
0 100 60∗ 70∗ 80∗ 70∗ 60∗
MEMPL, methanol extract of M. peltatus leaf. Values are the percentage animals showing a negative result; n = 10. ∗ P < 0.05 compared with control (Chi-square test).
Table 7 Preliminary phytoconstituents in MEMPL Serial No.
Phytoconstituents
Leaf extract of M. peltatus
1 2 3 4 5 6 7 8 9 10 11
Alkaloids Triterpenoids Steroids Amino Acids Flavonoids Gums Reducing sugars Tannins Saponins Fraction A Fraction B
+ (in traces) + + − + − + + + Ursolic acid -Sitosterol, minor fatty acids
−, Absence; +, presence.
Table 5 Effect of MEMPL on phenobarbitone sodium-induced sleeping time Experiment
Dose
Sleeping time (min)
Propylene glycol MEMPL
5 ml kg−1
64 70 82 119 114 75
Fraction A Fraction B
100 mg kg−1 200 mg kg−1 300 mg kg−1 50 mg kg−1 50 mg kg−1
± ± ± ± ± ±
0 2.0∗ 3.0∗ 3.0∗ 1.1∗ 2.0∗
MEMPL, methanol extract of M. peltatus leaf. Values are mean ± S.E., n = 10. ∗ P < 0.001 compared with control.
major group of compounds namely, fraction A (ursolic acid, triterpene), fraction B (-sitosterol and some fatty acid).
4. Discussion The results indicated that the MEMPL influences general behavioral profiles, as evidenced in the spontaneous activity, touch, sound and pain responses. The MEMPL possess significant analgesic activity compared with the standard drug
104
D. Chattopadhyay et al. / Journal of Ethnopharmacology 85 (2003) 99–105
pethidine in a dose-dependent manner. This activity may be due to its action on central nervous system like pethidine. M. peltatus leaf extract was reported to decrease the normal body temperature and yeast provoked elevation of body temperature significantly in a dose-dependent manner at 100–300 mg kg–1 doses (Chattopadhyay et al., 2002b). The extract significantly potentiated the phenobarbitone sodium-induced sleeping time, possibly through a CNS depressant action (Fastier et al., 1957) or a tranquilizing action (Mandal et al., 2001). The possible CNS activity of the methanolic extract, as well as the major fractions, was further investigated by other common psychopharmacological tests like the rotarod test, 30◦ inclined screen test and traction test. The reduction in exploratory behavior in animals treated with the methanolic crude extract or fractions A, and B of M. peltatus leaf is similar with the action of other CNS depressant agents. A significant lack in motor coordination and muscle relaxant activity was also noted in animals treated with the crude extract as well as with the fraction A. It has been reported that species of Mallotus (M. philippinensis, M. japonicus, M. repandus) contain flavonoids (mallotus A and B), terpenoids (mallotucin and malloripine; diterpenic lactones mallotucin C and D), saponins (rhamnoside crotoxigenin and coroglusagenine), cardinolipids, rottlerine and isorottlerine (The Wealth of India, 1985; Ambasta, 1992). But until recently, there are no reports on the chemistry or pharmacology of M. peltatus var acuminatus. For the first time, we have isolated two major compounds, ursolic acid (fraction A) and -sitosterol alongwith some fatty acids (fraction B). Contemporary scientific research revealed that the ursolic acid isolated from herbal sources have several pharmacological actions, like anticancerous (Kim, 1997), antiulcer, antihyperlipidemic, anti-inflammatory, hepatoprotective, antimicrobial and antiviral (Liu, 1995; Sattar et al., 1995; Zaletova et al., 1987; Chattopadhyay et al., 2001, 2002a). Ursolic acid isolated from several medicinal plants is found to inhibit human leukocyte elastase (Safayhi et al., 1997), 5-lipooxygenase and cyclooxygenase (Najid et al., 1992), concavalin A-induced histamine release (Tsuruga et al., 1991), and enzymes of arachidonate cascade (Diaz et al., 2000). The -sitosterol, on the other hand, reported to have anticancerous, antiulcer, antidiabetic, anti-inflammatory and antipyretic activities (Gupta et al., 1996; Pegel, 1980), and is helpful in controlling rheumatoid arthritis (Pegel, 1980). The present investigation for the first time, confirms that there is a moderate to strong degree of antipsychotic activity in the MEMPL, which appears to be due to the action of either tritepenes (fraction A) alone, or the combination of tritepenes and -sitosterol (fraction B) present in the methanol extract. In conclusion, the vernacular medicinal uses of M. peltatus leaf for mental tension and disturbance as well as to induce sleep is validated by our findings.
Acknowledgements The authors are grateful to the Department of Biotechnology, Government of India, for financial assistance through the project Grant No. BT/PRO 237/OSC 17/005/96. Thanks are due to Dr. Sreekumar, Senior Scientist, and the Officer In-Charge, Botanical Survey of India, Andaman & Nicobar Circle, Port Blair, Andamans for identification of the plant species. We express our gratitude to Dr. S.K. Bhattacharya, Director, National Institute of Cholera & Enteric Diseases, Kolkata and Officer In-Charge, ICMR Virus Unit Calcutta, for his constant encouragement, help and suggestions.
References Ambasta, S.P. (Ed.), 1992. The Useful Plants of India. Publications & Information Directorate (P & ID), CSIR, New Delhi, pp. 31–32. Asolkar, L.V., Kakkar, K.K., Chakre, O.J. (Eds.), 1992. Second Supplement to Glossary of Indian Medicinal Plants with Active Principles, Part 1 (A-K). Publications & Information Directorate, CSIR, New Delhi, pp. 51–52. Bhargava, N., 1983. Ethnobotanical studies of the tribes of Andaman and Nicobar Islands, India. I. Onge. Economic Botany 37, 110–119. Chakraborty, T., Vasudeva Rao, M.K., 1988. Ethnobotanical studies of the Shompen of Great Nicobar Islands. Journal of Economic and Taxonomic Botany 12 (1), 43–54. Chattopadhyay, D., Arunachalam, G., Mandal, A.B., Sur, T.K., Mandal, S.C., Bhattacharya, S.K., 2002a. Antimicrobial and antiinflammatory activity of folklore Mallotus peltatus leaf extract. Journal of Ethnopharmacology 82, 229–237. Chattopadhyay, D., Arunachalam, G., Mandal, A.B., Mandal, S.C., 2002b. Evaluation of antipyretic activity of leaf extract of Mallotus peltatus (Geist) Muell Arg. var acuminatus: a folk medicine. Phytomedicine 9, 727–730. Chattopadhyay, D., Maiti, K., Kundu, A.P., Chakrabarty, M.S., Bhadra, R., Mandal, S.C., Bhattacharya, S.K., 2001. Antimicrobial activity of Alstonia macrophylla: a folklore of Bay Islands. Journal of Ethnopharmacology 77, 49–55. Dagar, H.S., Dagar, J.C., 1991. Plant folk medicines among Nicobarese of Katchal Island, India. Economic Botany 45 (I), 114–119. Dandiya, P.C., Collumbine, H., 1959. Studies on Acorus calamus (III): some pharmacological action of the volatile oil. Journal of Pharmacology and Experimental Therapeutics 125, 353–359. Diaz, A.M., Abad, M.J., Fernandez, L., Recuero, C., Villaescusa, L., Silvan, A.M., Bermejo, P., 2000. In vitro anti-inflammatory activity of iridoids and triterpenoid compounds isolated from Phillyrea latifolia L. Biological & Pharmaceutical Bulletin 23 (11), 1307–1313. Dixit, V.K., Varma, K.C., 1976. Effects of essential oil of leaves of Blumea lacera DC on central nervous system. Indian Journal of Pharmacology 18, 7–11. Dorr, M., Stienberg, H., Tomkiewiez, M., Joyee, D., Porosolt, R.D., Summerfield, A., 1971. Persistence of dose related behaviour in mice. Nature 231, 121–123. Dunham, N.W., Miya, T.S., 1957. A note on simple apparatus for detecting neurological deficit in rats and mice. Journal of American Pharmacology 46, 208–209. Fastier, F.N., Spenden, R.N., Hendrieka, W., 1957. Prolongation of chloral hydrate sleeping time by 5-HT and by certain other drugs. British Journal of Pharmacology 12, 251–259. Ghosh, M.N. (Eds.), 1984. Fundamental of Experimental Pharmacology, 2nd ed. Scientific Book Agency, Calcutta, p. 153.
D. Chattopadhyay et al. / Journal of Ethnopharmacology 85 (2003) 99–105 Gupta, M.B., Nath, R., Srivastava, N., Shanker, K., Kishor, K., Bhargava, K.P., 1996. Anti-inflammatory and antipyretic activities of -sitosterol. International Journal of Immunopharmacology 18 (12), 693–700. Kim, K.-W., 1997. Anticancer activities of plant triterpenoids, ursolic acid and oleanoid acid. Journal of Korean Association and Cancer Prevention 2, 38–44. Litchfield, J.T., Wilcoxon, F., 1949. A simplified method of evaluating dose–effect experiments. Journal of Pharmacology and Experimental Therapeutics 96, 99–133. Liu, J., 1995. Pharmacology of oleanolic and ursolic acid. Journal of Ethnopharmacology 49, 57–68. Mandal, S.C., Dhara, A.K., Maiti, B.C., 2001. Studies of psychopharmacological activity of Andrographis paniculata extract. Phytotherapy Research 15, 253–256. Mukherjee, P.K., Saha, K., Balasubramanium, R., Pal, M., Saha, B.P., 1996. Studies on psychopharmacological effects of Nelumbo nucifera Gaertn. Rhizome extract. Journal of Ethnopharmacology 54, 63–67. Murugesan, T., Ghosh, L., Das, J., Pal, M., Saha, B.P., 1999. CNS activity of Jussiaea suffruticosa Linn. extract in rats and mice. Pharmacy and Pharmacology Communication 5, 663–666. Najid, A., Simon, A., Cook, J., Chable-Rabinobitch, H., Delage, C., Chulia, A., Riguad, M., 1992. Characterization of ursolic acid as a lipoxygenase and cyclooxygenase inhibitor using macrophages, platelets and differentiated HL 60 leukemic cells. FEBS Letter 299 (3), 213–217. Pegel, K.H., 1980. The importance of sitosterol and sitosterolin in human and animal nutrition. Planta Medica 39, 157–163. Plummer, D.I. (Ed.), 1985. An Introduction to Practical Biochemistry, 2nd ed. Tata Macgrow-Hill Publishing Co. Ltd., New Delhi, pp. 136, 143. Pollock, J.R.A., Stevens, R. (Eds.), 1965. Dictionary of Organic Compounds, vol. 5, 4th ed. Eyre & Spottiswoode Publishers Ltd., London, pp. 127–217. Randall, P., 1960. Studies of the neurological effects using simple experimental model. Journal of Pharmacology and Experimental Therapeutics 135, 543–545.
105
Rudzik, A.D., Hester, J.B., Tang, A.H., Staw, R.N., Friis, W. (Eds.), 1973. The Benzodiazepines. Raven Press, New York, pp. 285– 297. Rushton, R., Steinberg, H., Tinson, C., 1961. Modification of the effects of an amphetamine barbiturate mixture by the past experience of rats (Y-shaped runway). Nature 192, 533–535. Safayhi, H., Rall, B., Sailer, E., Ammon, H., 1997. Inhibition of boswellic acids of human leucocyte elastase. Journal of Pharmacology and Experimental Therapeutics 281 (10), 460–463. Sattar, A., Bankova, V., Kujumgiev, A., Galabov, A., Ignatova, A., Todorova, C., Papov, S., 1995. Chemical composition and biological activity of leaf exudates from some Lamiaceae plants. Pharmazie 50, 62– 68. Trease, G.E., Evans, W.C. (Eds.), 1983. Pharmacognosy, 12th ed. ELBS Publication, Baillier Tindall, East Bourne, pp. 344, 539. Tsuruga, T., Chun, Y., Ebizuka, Y., Sankawa, U., 1991. Biologically active constituents of Melaleuca leucadendron; inhibitors of induced histamine release from rat mast cells. Chemical and Pharmacological Bulletin 39 (12), 3276–3278. Turner, R.A. (Ed.), 1965. Screening Methods in Pharmacology. Academic Press, New York, pp. 26–35. Raw Materials, 1985. Publications & Information Directorate, CSIR, New Delhi, vols. I–XI, 1948–1976; revised and enlarged, vol. 1A, pp. 199–200. Wallis, T.E. (Ed.), 1985. Textbook of Pharmacognosy, 5th ed. CBS Publishers & Distributors, Delhi, pp. 15–20, 561–565. Woodson, R.F. (Ed.), 1987. Statistical Method for the Analysis of Bio-Medical Data, Probability and Mathematical Statistics. Wiley, Chichester, pp. 313–316. Zaletova, N., Shavlinskii, A., Tolkacchaev, O., Vichkanova, S., Fateeva, T., Krutikova, N., Yartseva, I., Klyuev, N., 1987. Preparation of some derivatives of ursolic acid and their antimicrobial activity. Chemical Abstracts 106, 18867e.