Evaluation of anxiolytic properties of Gotukola--(Centella asiatica) extracts and asiaticoside in rat behavioral models.Abstract
The ayurvedic medicinal plant Gotukola (Centella asiatica Centella asiatica is a small herbaceous annual plant of the family Apiaceae, the carrot and dill family, native to northern Australia, New Guinea, Melanesia, Malaysia, Iran] and other parts of Asia. ) was evaluated for its anxiolytic anxiolytic /anx·io·lyt·ic/ (ang?ze-o-lit´ik)
2. an antianxiety agent.
A drug that relieves anxiety. properties. Specifically, this study assessed the effects of: Gotukola plant materials of different genotypic genotypic
emanating from or pertaining to genotype.
selection of breeding stock on the basis of known inherited characteristics. origin; hexane hexane /hex·ane/ (hek´san) a saturated hydrogen obtained by distillation from petroleum.
n. , ethyl acetate ethyl acetate
A colorless volatile flammable liquid, CH3COOC2H5, used in perfumes, flavorings, lacquers, pharmaceuticals, and rayon and as a general solvent. and methanol extracts of Gotukola; and asiaticoside, a triterpenic compound isolated from Gotukola. Various paradigms were used to assess the anxiolytic activity, including the elevated plus maze (EPM EPM
equine protozoal myeloencephalitis. ), open field, social interaction, locomotor activity Locomotor activity (LMA) refers to the movement from place to place. In psychopharmacology, locomotor activity of lab animals is often monitored to assess the behavioural effects of these drugs. , punished drinking (Vogel) and novel cage tests. The EPM test revealed that Gotukola, its methanol and ethyl acetate extracts as well as the pure asiaticoside, imparted anxiolytic activity. Furthermore, the asiaticoside did not affect locomotor activity, suggesting these compounds do not have sedative sedative, any of a variety of drugs that relieve anxiety. Most sedatives act as mild depressants of the nervous system, lessening general nervous activity or reducing the irritability or activity of a specific organ. effects in rodents.
[c] 2006 Elsevier GmbH. All rights reserved.
Keywords: Gotukola (Centella asiatica); Natural health product; Asiaticoside; Anxiety; Rat trial; Elevated plus maze; Open field; Social interaction; Locomotor activity; Vogel; Novel cage
Gotukola (Centella asiatica L. Apiaceae) is a psychoactive medicinal plant that has been used for centuries in Ayurvedic medicine Ayurvedic Medicine Definition
Ayurvedic medicine is a system of healing that originated in ancient India. In Sanskrit, ayur means life or living, and veda to alleviate symptoms of anxiety and to promote a deep state of relaxation and mental calmness during meditation practices. Recent investigations using human and animal models of anxiety have confirmed that Gotukola does indeed possess anxiolytic activity. Bradwejn et al. (2000) reported that a single 12g dose of Gotukola administered orally was more effective than placebo in decreasing acoustic startle response Noun 1. startle response - a complicated involuntary reaction to a sudden unexpected stimulus (especially a loud noise); involves flexion of most skeletal muscles and a variety of visceral reactions
startle reaction in healthy humans. This effect was most pronounced 60min after treatment. In animals, Gotukola increases pentobarbitone-induced sleeping time and decreases immobility immobility
standing still and disinclined to move, as in an animal suddenly blinded; responds to other stimuli unless immobility is part of a dummy syndrome when all stimuli are ignored. in the forced swim test (Sakina and Dandiya, 1990). Gotukola also elicits anti-anxiety effects in the elevated plus maze (Lucia et al., 1997) and an aqueous extract an extract obtained from a vegetable substance by steeping it in water.
See also: Aqueous of Gotukola was reported to have cognitive-enhancing as well as antioxidant antioxidant, substance that prevents or slows the breakdown of another substance by oxygen. Synthetic and natural antioxidants are used to slow the deterioration of gasoline and rubber, and such antioxidants as vitamin C (ascorbic acid), butylated hydroxytoluene effects in rats (Kumar and Gupta, 2002).
The most prominent group of biologically active compounds isolated from Gotukola is the terpenes terpenes (terˑ·pēnz),
n.pl a large-sized group of unsaturated hydrocarbons with the empirical formula (C5H8)n. (Shukla et al., 1999a). Asiaticoside is the most abundant triterpene triterpene
plant toxins, e.g. lantadenes A, B, found in Lantana camara, icterogenins A, B, C, found in Lippia spp. Called also triterpene acids.
see triterpene (above). glycoside, which is effective in wound healing wound healing Physiology The repair of a wound Steps Inflammation, repair and closure, remodeling, final healing; repair of incisions may be either simple–'clean' wounds with little loss of tissue heal by 'primary intention', or 'dirty' wounds heal by and apparently acts by enhancing the induction of antioxidant levels at an early stage of wound healing (Shukla et al., 1999b). Asiaticoside is transformed into its aglycone aglycone /agly·cone/ (a-gli´kon) aglycon.
the noncarbohydrate portion of a glycoside molecule. asiatic acid in vivo by hydrolysis hydrolysis (hīdrŏl`ĭsĭs), chemical reaction of a compound with water, usually resulting in the formation of one or more new compounds. . Several derivatives of asiaticoside (Inhee et al., 1999) and asiatic acid (Sang-sup et al., 2000) were found to show protective effect against beta amyloid-induced neurotoxicity neurotoxicity /neu·ro·tox·ic·i·ty/ (noor?o-tok-sis´it-e) the quality of exerting a destructive or poisonous effect upon nerve tissue. associated with the dementia of Alzheimer's disease Alzheimer's disease (ăls`hī'mərz, ôls–), degenerative disease of nerve cells in the cerebral cortex that leads to atrophy of the brain and senile dementia. .
In the Bradwejn et al. (2000) study, the effective anxiolytic dose of Gotukola in humans was estimated at 12 g of crude leaf material per subject. Administration of such large amounts of plant material is awkward and impractical. The first objective of the present study was to identify more active plant genotypes as well as the active fraction(s) and compounds from the plant. The second was to identify the efficacy of these compounds across a variety of paradigms capable of detecting anxiolytic activity, including the elevated plus maze, open field test, social interaction test, locomotor activity, Vogel test and novel environment test.
Materials and methods
Test protocols were reviewed and approved by the University of Ottawa
1. As stated or indicated by; on the authority of: according to historians.
2. In keeping with: according to instructions.
3. guidelines of the Canadian Council Canadian Council may refer to:
River, eastern Massachusetts, U.S. The longest river wholly in the state, it flows into Boston Bay after a course of about 80 mi (130 km). Navigable for about 7 mi (11 km), its estuary separates the cities of Boston and Cambridge. Canada Inc. (St-Constant, QC) a week before the tests. Upon arrival rats were housed individually in standard rat cages measuring 45 x 24 x 20 cm (Lab Products, Pennsylvania, USA) and had ad libitum ad libitum
ad libitum feeding
food available at all times with the quantity and frequency of consumption being the free choice of the animal. access to standard Purina Rat Chow (Purina; code 5012) and tap water. The subjects were maintained in a tightly controlled environment (ventilation (100% fresh-air 20 exchanges/hour), lighting (12 h light-dark cycle; 7.00 a.m.-7.00 p.m.) and room temperature (21 [degrees]C). Bedding (Prochips; maple or birch hardwood chips) was changed once a week. During the habituation habituation
Reduction of an animal's behavioral response to a stimulus, as a result of a lack of reinforcement during continual exposure to the stimulus. Habituation is usually considered a form of learning in which behaviours not needed are eliminated. period of at least 5 days after arrival, rats were familiarized with the researcher(s).
Drug administration and testing of rats were conducted in a sound attenuated room. A mild detergent, (Quatsyl; 8 ml/1 water, Pharmacia & Upjohn Animal Health, Orangeville, Ontario) was used to clean the cages between tests.
Apparatus and test procedures
Elevated plus maze (EPM)
The EPM evokes conflict between the need to explore the novel area and the need to avoid more vulnerable (or aversive aversive /aver·sive/ (ah-ver´siv) characterized by or giving rise to avoidance; noxious.
adj. ) areas of the EPM (heights and open spaces). This maze comprises two open arms (or planks) transected by two perpendicularly opposing closed arms (or alleys with 40 cm high walls). The arms measure 50 cm in length and are 10 cm wide; the whole apparatus is elevated 50 cm off the floor with a stand. The floor of the EPM is made of a black rubberized runway and all interior walls are made of black Plexiglas. The apparatus was surrounded by a black curtain to minimize distractions. Light levels at the center, open- and closed arms of the EPM apparatus were 35, 40 and 4 lux, respectively. A closed circuit camera positioned above the maze permitted remote observation and scoring of animal behavior.
Following drug administration, rats were returned to their home cages for designated periods, and were tested in the open field arena for 5 min, just before placement onto the EPM. They were placed in the central, open square facing the closed arm and monitored for 5 min. The behaviors scored included: number of entries and time spent on the open arms (all four paws in open arm); time spent in the closed arms; number of occurrences and time spent in protected head dips (the animal dipping its head over the sides of open arm while part of the body is within the closed arm); and number of unprotected head dips (made from the open arm without contact with walls of closed arms). After 5 min of testing, rats were returned to their home cages.
Open field test
In this test, the aversion to the central zone (or vulnerable area) of an arena is used as an index of anxiety levels. The open-field apparatus constituted of a rectangular plexiglas arena measuring 60 x 60 cm with 35 cm high walls. The floor was marked with lines that divided it into 36 squares (10 x 10 cm). The squares immediately adjacent to the walls of the test arena constitute the 'safer' peripheral zone, whereas the inner or more centrally positioned squares were identified as the central (or vulnerable) zone. The behavior was monitored via a closed-circuit video camera mounted on the ceiling. The test apparatus was surrounded by black curtains to minimize undue distraction. Light levels at the center and perimeter of the open field test arena were 22 and 12 lux, respectively. The test was initiated by placing the rat into the center of the arena. Over a period of 5 min, the number of squares crossed and time spent in the center and the perimeter was determined.
Social interaction test
In this test, the amount of time a pair of rats spend socially interacting with one another is thought to reflect the level of anxiety in these subjects. The duration of social interaction decreases with increased anxiety. Rats were placed individually in the test arena for 7-min familiarization session on two consecutive days. During the test day, two randomly selected rats were administered the drug and placed in adjacent cages in the waiting area of the test room. One hour later, they were introduced together into the center of the test arena. Social interaction was observed remotely for 7min, via the video camera. The number of occurrences of social interactions (sniffing, following, and grooming the partner) and time spent in them were scored.
In order to monitor general locomotor activity, the distance traversed by the rats in their home cage was monitored via a computerized infrared tracking device. This equipment permitted the assessment of locomotor activity, by tracking the number of zones (6 per cage) crossed and the total distance traversed. Drugs were administered at 10 a.m. and the data collection initiated at II a.m. lasted for 22 h (until 9 a.m. the next day). During the light phase (7 a.m. 7 p.m.), the light level in the test cages was 90 lux, followed by a dark phase (7 p.m.-7 a.m.).
Vogel test (punished-drinking conflict)
In this test, thirsty (water-deprived) rats are given access to water, however each fifth lick is accompanied with a mild shock (delivered through the drinking spout). The lower the level of anxiety, the greater the number of punished licks accepted. The Vogel cage (Habitest Operant operant /op·er·ant/ (op´er-ant) in psychology, any response that is not elicited by specific external stimuli but that recurs at a given rate in a particular set of circumstances.
adj. Cage H 13-16, Coulbourn Instruments, Allentown, PA, USA) is made of Plexiglas and measures 30 x 25 x 30 cm. The box has a floor with metal rods spaced 2 cm apart and is has a water spout tube connected to an external shocking device. The water bottle is placed on the outside of the test box (about 3 cm above the grid floor) and spout protrudes 2 cm into the cage. A mild shock (0.1 or 0.4 mA) was delivered through the spout on every fifth lick. The number of licks emitted was tracked electronically over the 10-min test, via an optical beam (that is interrupted by the tongue when it contacts the spout). Rats were previously trained for two consecutive days to locate and drink from the water spout, in the absence of shock. They were then deprived of water for 22 h and randomly assigned to various test groups. The ambient light level was 90-100 lux.
Novelty induced feeding suppression test suppression test A test or assay–eg, dexamethasone suppression test, used to determine whether a substance–hormone or protein being produced in excess is under the control of regulating or releasing factor(s), and therefore responsive to a feedback
When rats are presented with a familiar palatable snack in their home cage, they readily approach and consume it. However, when the same snack is presented in a novel environment, the rat's latency to approach the snack is markedly increased, and the amount consumed is decreased, effects reversible by anxiolytic drugs (Merali et al., 2003). Rats were given daily (20min) access to a palatable snack (Honey Maid brand graham crackers) in their home cage for eight consecutive days. On the test day, rats were treated with the drug(s) and transferred into a novel cage and presented with the same palatable snack. The latency to initiate snack consumption and the amount consumed were measured. The ambient light level was 90-100 lux.
Ayurveda recommends mixing Gotukola with milk before its administration in order to achieve the intended psychoactive effects (Sushruta, ~600BC). Dry powder Dry Powder
A slang term for cash reserves kept on hand to cover future obligations.
For example, if a venture capitalist expects bad times in the IPO markets you might hear him say something like, "we want to keep enough dry powder around to keep funding our and fresh leaf extracts of Gotukola were sonicated for 15min in 50% sweetened condensed milk condensed milk: see milk. solution (Sunfresh Ltd., Toronto, Ontario) or distilled water Noun 1. distilled water - water that has been purified by distillation
H2O, water - binary compound that occurs at room temperature as a clear colorless odorless tasteless liquid; freezes into ice below 0 degrees centigrade and boils above 100 degrees centigrade; , to make suspensions. The treatments were given orally (p.o.) using a gavage gavage /ga·vage/ (gah-vahzh´) [Fr.]
1. forced feeding, especially through a tube passed into the stomach.
1. tube (gage 18, length 5 cm). Asiaticoside was sonicated in peanut oil peanut oil
The oil pressed from peanuts, used for cooking, in soaps, and as a solvent for pharmaceutical preparations.
Noun 1. to make suspensions for intra-peritoneal (i.p.) administration. The volume of drug or vehicle (control) was approximately 2 ml/rat or less for p. o. and less than 1 ml/kg body wt. in the case of intraperitoneal (i.p.) injections. Rats were habituated to drug administration modalities for two consecutive days prior to the tests. Different dosages and post-treatment test intervals were assessed (see descriptions of particular tests). Immediately after drug administration, rats (in home cages) were brought into the test room and placed in the holding area adjacent to the test apparatus.
Dosage, post-drug interval and treatment administration
Effects of whole plant materials from different Gotukola products
Two high-quality commercial Gotukola-natural health products (NHP NHP Non-Human Primate
NHP Natural Health Product
NHP Nevada Highway Patrol
NHP National Historic Park
NHP Nottingham Health Profile
NHP National Health Plan
NHP Nursing Home Placement
NHP Nominal Horsepower
NHP Not-Hot Plug (server) ) containing relatively higher or lower concentrations of asiaticoside were selected for this study. The first product was dried leaf material grown from a genotype genotype (jēn`ətīp'): see genetics.
Genetic makeup of an organism. The genotype determines the hereditary potentials and limitations of an individual. from the Indian subcontinent (NHP1). The second commercial product was a genotype from Madagascar (NHP2) with high triterpene content. Reference materials have been deposited in the University of Ottawa Herbarium herbarium, collection of dried and mounted plant specimens used in systematic botany. To preserve their form and color, plants collected in the field are spread flat in sheets of newsprint and dried, usually in a plant press, between blotters or absorbent paper. . High Performance Liquid Chromatography High-performance liquid chromatography (HPLC) is a form of column chromatography used frequently in biochemistry and analytical chemistry. It is also sometimes referred to as high-pressure liquid chromatography. (HPLC HPLC high-performance liquid chromatography.
high performance liquid chromatography.
HPLC High-performance liquid chromatography Lab instrumentation A highly sensitive analytic method in which analytes are placed ) analysis of asiaticoside and asiatic acid was undertaken using a validated method reported elsewhere (Wijeweera, 2003). Separations were achieved using a YMS YMS Yardley-Makefield Soccer (Pennsylvania)
YMS Yard Management System (system for managing container terminal yards)
YMS Yield Management System
YMS Young Men's Survey 3-[micro]m ODS (Operational Data Store) A database designed for queries on transactional data. An ODS is often an interim or staging area for a data warehouse, but differs in that its contents are updated in the course of business, whereas a data warehouse contains static data. C18 column (2 x 100 mm) (Waters, Mississauga ON), flow 0.2ml/min, with a 0.3% formate/MeCN gradient and detection at 205 nm. The gradient was 10-80% acetonitrile acetonitrile /ac·e·to·ni·trile/ (as?e-to-ni´tril) a colorless liquid with an etherlike odor used as an extractant, solvent, and intermediate; ingestion or inhalation yields cyanide as a metabolic product. in 8min, 80 100 MeCN for 2min, hold 2.5 min, 100-10% MeCN for 2.5 min. Plant material (1 g) was defatted defatted
1. fat is removed from the tissue by fat solvents.
2. deprived of fat as a food. in hexane (2 x 20 ml, shaker 1 h) and then extracted in MeOH (3 x 20 ml, sonicator 15 min, shaker 6h). The filtered extracts were concentrated in vacuo and redissolved in 10 ml MeOH before HPLC analysis. Quantification was performed by comparison to an authentic standard of asiaticoside.
The following test parameters were used for the EPM test. The NHP1 and NHP2 products were tested at 200mg/kg body wt. dosage, using distilled water as vehicle (control) and 1 h post-treatment interval (Test Al). The same test was then repeated using a higher dose (500 mg/kg body wt.) and a longer post-treatment interval (2h; Test A2).
For the Novel Cage Test, the NW and SM products were tested at 200 mg/kg body wt. dosage with distilled water as the vehicle and 1 h post-treatment interval (Test A3).
Effects of Gotukola-extracts of different polarity
The aerial parts of the tissue-culture-propagated Gotukola plants grown in the university greenhouse were cleaned and oven dried at 40 [degrees]C for 24 h and crushed into powder. Gotukola powder (128 g) obtained from 750 g of fresh plant material was used for extractions. The powder was first extracted with hexane: (41, sonicated for 15min, magnetic shaker 6h) x 3. The hexane-insoluble material was then extracted with ethyl acetate: (41, sonicated for 15min, magnetic shaker 6h) x 3. From the hexane- and ethyl ethyl (ĕth`əl), CH3CH2, organic free radical or alkyl group derived from ethane by removing one hydrogen atom. acetate-insoluble residue, the final extract was made with methanol: (41, sonicated for 15min, magnetic shaker 6h) x 3. The extracts were filtered and concentrated in vacuo. The extracted fractions (hexane--2.12g, ethyl acetate--1.11 g, methanol--30.47 g) were then dissolved in 50 ml of 50% condensed milk.
The hexane extract (212 mg/kg body wt.), ethyl acetate extract (111 mg/kg body wt.) and the methanol extracts (3047 mg/kg body wt.) were administered p.o in 50% condensed milk, and the animals tested on the EPM, 2h following treatment.
Effects of the pure asiaticoside compound
A pure compound of Gotukola, asiaticoside, was purchased from Indofine Chemicals, Somerville, NJ, USA. The study was conducted with different test conditions as summarized in Table 2. For the elevated plus maze test (C1), in the initial experiment, the asiaticoside was administered at doses of 1 and 3mg/kg body wt. (in peanut oil), and rats tested 1 h later (Experiment C1). The next experiment was a dose-response study where 3, 5 and 10 mg/kg body wt. of the compound were tested, 1 h after administration (Experiment C2). For the open field test, asiaticoside was dosed at 3, 5 and 10 mg/kg body wt. (in peanut oil), 1 h following administration (Experiment C3). For the social interaction test, asiaticoside was dosed at 1 and 3 mg/kg body wt. (in peanut oil) and tested 1 h following administration (Experiment C4). In the next experiment, the asiaticoside was tested at 1 and 3 mg/kg body wt. dosages and tested 4 h following treatment (Experiment C5). In the locomotor activity test, rats were administered the asiaticoside (1 and 3 mg/kg body wt. in peanut oil) and locomotor activity monitored on an hour-by-hour basis, for 23 h (Experiment C6). For the Vogel test, rats were treated with asiaticoside (1 and 3 mg/kg body wt. in peanut oil) and tested in the punished-drinking (Vogel) paradigm, 1 h following administration. The shock level at the drinking spout was set at 0.4 mA (Experiment C7). In the next experiment, The effects on punished drinking were further assessed using a higher dose of the asiaticoside (5 mg/kg body wt. in peanut oil) and a lower shock intensity (0.1 mA). Once again, rats were tested 1 h following drug administration (Experiment C8). In the next set of experiments, the effects of asiaticoside (5 mg/kg body wt.) were assessed at various time periods following drug administration (0.5, 1 and 2h). The shock intensity at the spout was set at 0.1 mA.
The main focus of the study was to investigate the anxiolytic activity of different phytochemical phy·to·chem·i·cal
A nonnutritive bioactive plant substance, such as a flavonoid or carotenoid, considered to have a beneficial effect on human health. components of Gotukola, compared to the controls. Based on the prior assumptions, post hoc post hoc
adv. & adj.
In or of the form of an argument in which one event is asserted to be the cause of a later event simply by virtue of having happened earlier: analyses were carried out using the students t-test. Data from rats that fell off the EPM were omitted from statistical analyses.
Results and discussion
The effects of high- and low-asiaticoside commercial natural health products
A pronounced anxiolytic effect was observed in rats administered the higher dose (500 mg/kg body wt.) of either of the two commercial Gotukola products, as compared to the control vehicle (see Table 1). At a lower dose (200 mg/kg body wt.), significant anxiolytic effects were not detected with either of the two commercial NHPs (data not shown). These results are consistent with the clinical observations in healthy volunteers, where significant anxiolytic effects were found with NHP1 at the relatively high doses (12g/person).
At the 500 mg/kg body wt. dose, significant anxiolytic activity of NHP1 was observed in five out of seven measures assessed (at p < 0.001 level); Compared to control, NHP1 significantly: (a) increased duration of time spent on the open arms of the EPM; (b) decreased duration on the closed arms; (c) increased number of protective head dips; (d) increased time spent in the protected head dip activity; (e) increased number of unprotected head dips. The behavioral profile of NHP2 was similar to that of NHP1, however, the number of open arm entries was also significantly increased. In terms of the magnitude of change, the greatest effects were seen in the time spent in open arms and the number of unprotected head dips.
Although NHP2 appeared to show greater anxiolytic activity than NHP1, the differences were not statistically significant. The NHP2 product had a higher triterpene content (asiaticoside and asiatic acid) as compared to the NHP1 product. As revealed by the HPLC analysis, NW and SM contained 0.37% and 2.40% of asiaticoside (dry wt. basis), respectively.
Experiment 2. The effects of Gotukola extracts of different polarity
The extraction was undertaken with Gotukola, grown in the greenhouse, freshly harvested and immediately dried, to ensure that the starting material was authentic and unaltered by any commercial process or storage. The methanol extract represented the most abundant fraction after removal of the solvent (23.8% dry wt.), followed by the hexane (1.66% dry wt.) and ethyl acetate (0.87% d.w.) fractions (Table 2). As revealed by HPLC, the asiaticoside concentration increased in the order hexane, ethyl acetate and methanol extract, respectively, as might be expected due to the oligosaccharide oligosaccharide: see carbohydrate.
Any carbohydrate with a few (between 3 and about 6 to 10) units of simple sugars (monosaccharides). A wide variety of oligosaccharides are made by partially breaking down polysaccharides. tail in asiaticoside. The more lipophilic lipophilic,
adj/n the ability to dissolve or attach to lipids.
adj 1. showing a marked attraction to, or solubility in, lipids.
2. aglycone, asiatic acid was most abundant in the ethyl acetate fraction.
The behavioral tests (Table 3) indicated that administration of the dried hexane extract failed to alter behavior significantly, but the ethyl acetate and methanol extracts both showed some anxiolytic activity. In the methanol extract group, the number of closed arm entries was significantly reduced and the time spent in protected head dips was significantly increased. In the ethyl acetate extract group, the number of protected head dips and the time engaged in this activity were significantly increased.
The result of this test are a clear indication of the presence of anxiolytic principles of dried residue in the ethyl acetate and methanol fractions of Gotukola and also suggest that the hexane fraction is devoid of pharmacological activity as an anxiolytic agent. The bioactivity clearly lies in fractions with significant triterpene content, and especially the methanol fraction.
Experiment 3. Effects of pure asiaticoside
Elevated plus maze (Experiment 3a and b).
The first set of experiments assessing the effects of various doses of the asiaticoside (ranging from 1 to 3 mg/kg body wt., Experiment 3a) were based on results obtained with NHP1 at 500 mg/kg body wt., which was estimated to contain between 1 and 2 mg/kg body wt. of the asiaticoside (1.85 mg/kg body wt.). There was clear evidence of anxiolytic effects of the pure compound (Table 4). There was a significant difference between control and both asiaticoside 1 and 3 mg/kg body wt. drug groups for number of closed arm entries and the time spent on the closed arm. In addition, the number of unprotected head dips was significantly increased by the 3 mg/kg body wt. dose (p<0.05). In this study, there was clear evidence of dose-response effects in parameters.
In a second trial, a higher dose-response study (0, 3, 5, 10 mg/kg body wt.) comparable to NHP2 delivery of asiaticoside (12 mg/kg body wt.) was undertaken (Table 5). It showed significant (p<0.05) anxiolytic activity at all three doses tested, as reflected by increased number of open arm entries, (b) increased time spent on open arm, (c) reduced time spent on the closed arm, and (d) increased number of protected head dips.
At the higher doses, more parameters were significantly affected. The amount of time engaged in protected head dip activity was significantly increased at the 5 mg/kg body wt. dose. At the highest dose, the number of unprotected head dips was significantly increased. This test did not indicate a clear dose-dependent relationship, and in fact the anxiolytic effect appeared to have reached a plateau or maximal effect. Still, the magnitude of the change in parameters was large. The number of open arm entries and time spent on the open arms doubled in the three treated groups as compared to controls, while the time and number of protected head dips in the three treated groups was at least 150% of that seen in controls.
To get a better picture of the dose response effect, data from the low- and high-dose experiments were combined (Fig. 1) for a responsive variable, the time in the open arm. Clearly, there is very steep concentration dependence in the behavioral effect followed by a rapid saturation. Steep slopes in pharmacological responses often reflect specific binding of the pharmacological agent to relevant receptors. This steep slope was also clearly evident with the NHP products since they were inactive at the 200 mg/kg body wt. dose and active at the 500 mg/kg body wt. dose. In addition, the lack of difference between NHP1 and NHP2 may be due to rapid saturation of the effect for both products at 500 mg/kg body wt.
Open field test (C3: 3-10 mg/kg body wt. dose-response study)
The open field test (Table 6) revealed that the rats spent significantly more time at the center of the arena when treated with the asiaticoside (3 mg/kg body wt.) (35.9 s) and 5 mg/kg body wt. (41.3 s), compared to the controls (24.8 s). This observation further supports the view that this asiaticoside imparts anxiolytic activity. As in the EPM test, a dose-dependent response was not observed in this dose range.
Social interaction test (C4, C5: 1-3 mg/kg body wt. dose--response study)
In this test (Table 7), a significant reduction in the number of non-interaction activity was observed with the 1 mg/kg body wt. dose (29.8 s) compared to the control (35.0 s). Although this observation suggests anxiolytic activity of asiaticoside, the significant increase in the non-interaction time with the 3 mg/kg body wt. dosage (251.7 s), compared to the control (207.5 s) does not support such an activity. In the test C5, no significant difference among drug groups compared to the control group was observed after 4 h of post-treatment (data not shown.) It seems that 4 h after the experiments is too late to still exhibit any possible biological activity under the tested dosage.
[FIGURE 1 OMITTED]
Locomotor activity test (C6)
During the 22 h of the test period, no significant difference was observed between the control and the asiaticoside (1 and 3 mg/kg body wt.) treated groups, in the number of squares crossed or total distance traversed (data not shown). Asiaticoside at 1 and 3 mg/kg body wt. dosages did not affect the ultradian locomotor activity, an indication that asiaticoside does not have sedative effects (Fig. 2).
Vogel test (C7, C8, C9)
No significant group differences in the number of punished licks endured was apparent in the groups treated with the asiaticoside (1 and 3 mg/kg body wt.) as compared to the control, after a 1-h post-drug interval (data not shown). No significant difference was observed in test C8 in the response of the asiaticoside (5 mg/kg body wt.)-treated group and control group after a 1-h post-drug interval (data not shown). Although there was a trend towards an increased number of punished licks endured by the treated rats in the 0.5 h post-drug interval (587.4), in the 1 h (795.2) and 2 h (602.1) post-drug interval groups, the difference was not statistically significant (data not shown). Compared to the C7 test, in which a shock of 0.4 mA was used, a relatively much lower shock of 0.1 mA was applied in the C8 and C9 tests. Other than the increase in the asiaticoside dosage level, the low shock applied for tests C8 and C9 may have had an impact on the relatively higher number of licks compared to the C7 test.
This study, deploying several animal models of anxiety, provides strong support to the ayurvedic claim that Gotukola has anxiolytic activity. The data reported herein provide unequivocal evidence for the anxiolytic activity of not only the crude plant material, but also the specific organic extracts and at least one bioactive principle. The findings also support and extend some of the previous preliminary studies on the anxiolytic profile of Gotukola, by Sakina and Dandiya (1990) and Diwan Noun 1. diwan - a Muslim council of state
privy council - an advisory council to a ruler (especially to the British Crown)
2. diwan - a collection of Persian or Arabic poems (usually by one author)
divan et al. (1991).
[FIGURE 2 OMITTED]
Our results suggest that this anxiolytic activity may be attributable in part to triterpene rich fractions within the plant extracts. Asiaticoside is clearly one of the active triterpenes, and is found in the plant in the largest amount, but there may be other active principles and some synergy between them and the whole plant activity may be important. It is probable that the other terpenes contribute to activity and these should be tested as well.
The identification of terpene-rich fractions and asiaticoside as active principles provides a method for producing a more manageable (i.e., smaller) dosage to replace the large 12 g dry leaf dose used in previous clinical work. If the pure active principle were used, Fig. 1 suggests a saturation of the effect at a dose 3 mg/kg body wt. This corresponds to a 180-mg tablet for a 60-kg individual, a very manageable size. If consumer preference for natural plant extracts is considered, two capsules of 500 mg each would deliver the required dose for an extract standardized to 20% asiaticoside.
This study was funded by grants from the International Development Research Centre and the Natural Sciences and Engineering Research Council The Natural Sciences and Engineering Research Council (NSERC) is a Canadian government division that provides grants for research in the natural sciences and in engineering. In 2004-2005, it will invest CAD $850 million in university-based research and training. of Canada.
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1. starchlike; amylaceous.
2. the pathologic, extracellular, waxy, amorphous substance deposited in amyloidosis, being composed of fibrils in bundles or in a meshwork of polypeptide (A-[beta])-induced neurotoxicity. Bioorg. Med. Chem. Lett. 10, 119-121.
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Sushruta, 1991. ~600 BC. Sushruta Samhita Sushruta Samhita (sōō·shrōōˑ·t , fourth ed. Chowkhamba Press, Varanasi, India (English translation of the original Sanskrit text by Bhishagratna, K.K. in 1907, 1911, 1916; 3 volumes).
Wijeweera, P., 2003. Phytochemical basis for the anxiolytic activity of the ayurvedic medicinal plant, Centella asiatica. M.Sc. Thesis, University of Ottawa.
P. Wijeweera (a,d), J.T. Arnason (a,*), D. Koszycki (b), Z. Merali (b,c)
(a) Ottawa-Carleton Institute of Biology The Institute of Biology (IoB) is a professional body for biologists, primarily those working in the United Kingdom. Membership currently stands around 14,000. It was founded in 1950, received a Royal Charter in 1979 and holds charitable status. , University of Ottawa, Canada
(b) University of Ottawa Institute of Mental Health Research, Royal Ottawa Hospital, Canada
(c) Department of Psychology, University of Ottawa, Ottawa, Canada
(d) Natural Health Products Directorate The Natural Health Products Directorate (NHPD) is the division of the Health Products and Food Branch of Health Canada that is responsible for implementation of the Natural Health Product Regulations, including Good Manufacturing Practices, for Natural Health Products for sale in , Health Canada Health Canada (French: Santé Canada) is the department of the government of Canada with responsibility for national public health.
Health Canada's goal is to improve Canadian life by improving Canadian longevity, lifestyle and use of public healthcare. , Ottawa, Canada
*Corresponding author. Tel.: + 1 613 562 5262; fax: + 1 613 562 5765.
E-mail address: email@example.com (J.T. Arnason).
Table 1. Comparison of different Gotukola NHPs based on performance on the elevated plus maze: NHP1 and NHP2 (at 500 mg/kg body wt. dosage) are compared with the control (distilled water), after 2 h post-drug interval Treatment #o.a. Time o.a. (s) Control n = 10 2.2 [+ or -] 0.9 24.1 [+ or -] 9.7 NHP1 n = 10 3.7 [+ or -] 0.6 62.4 [+ or -] 10.8 (a) NHP2 n = 10 4.8 [+ or -] 0.7 (a) 66.0 [+ or -] 9.3 (a) Treatment #c.a. Time c.a. (s) Control n = 10 22.6 [+ or -] 11.2 208.9 [+ or -] 13.1 NHP1 n = 10 9.7 [+ or -] 1.2 143.7 [+ or -] 18.5 (a) NHP2 n = 10 10.2 [+ or -] .5 136.7 [+ or -] 10.7 (a) Treatment #phd Time phd (s) Control n = 10 12.8 [+ or -] 1.4 45.6 [+ or -] 6.7 NHP1 n = 10 18.6 [+ or -] 1.6 (a) 77.9 [+ or -] 9.4 (a) NHP2 n = 10 20.9 [+ or -] 1.2 (a) 86.0 [+ or -] 5.6 (a) Treatment #uphd Control n = 10 10.6 [+ or -] 4.0 NHP1 n = 10 29.4 [+ or -] 5.6 (a) NHP2 n = 10 33.7 [+ or -] 4.0 (a) All values represent the group mean [+ or -] s.e.m. (n = 10). #o.a. -- number of open arm entries, time o.a. -- time spent on open arm, #c.a. -- number of closed arm entries, time c.a. -- time spent on closed arm, #phd -- number of protected head dips. time phd - time spent on protected head dips, #uphd -- number of unprotected head dips. (a) p < 0.001 vs. control, t-test. Table 2. Fresh weight of Gotukola and yields of dried materials, hexane, ethyl acetate and methanol fractions Fresh materials Dried materials Hexane extract (a) Weight (g) 750 128 2.12 Yield (%) (b) 1.66 Asiaticoside (%) 0.01 Asiatic acid (%) 0.31 Ethyl acetate extract (a) Methanol extract (a) Weight (g) 1.11 30.5 Yield (%) (b) 0.87 23.8 Asiaticoside (%) 0.60 4.3 Asiatic acid (%) 1.3 0.61 Asiaticoside content and asiatic acid content of the three fractions is given. (a) After removal of solvent. (b) As a percentage of dry weight of whole plant materials. Table 3. Comparison of plus maze performance of rats treated with Gotukola hexane, ethyl acetate and methanol extracts with control (50% condensed milk), after 2h post-drug interval Treatment #o.a. Time o.a. (s) #c.a. Control 3.9 [+ or -] 0.8 51.2 [+ or -] 12.8 12.1 [+ or -] 1.0 (condensed milk) (n = 10) Hexane (n = 10) 4.3 [+ or -] 1.0 68.1 [+ or -] 17.2 11.0 [+ or -] 0.6 EtOAc (n = 9) 2.9 [+ or -] 1.1 38.4 [+ or -] 15.0 10.0 [+ or -] 0.6 MeOH (n = 11) 3.3 [+ or -] 0.8 41.4 [+ or -] 11.2 9.1 [+ or -] 0.9 (a) Treatment Time c.a. (s) #phd Control 190.8 [+ or -] 16.3 11.3 [+ or -] 1.5 (condensed milk) (n = 10) Hexane (n = 10) 166.5 [+ or -] 14.5 13.5 [+ or -] 1.0 EtOAc (n = 9) 168.9 [+ or -] 16.3 17.7 [+ or -] 1.2 (a) MeOH (n = 11) 174.5 [+ or -] 16.2 15.7 [+ or -] 1.7 Treatment Time phd (s) #uphd Control 36.3 [+ or -] 5.3 21.5 [+ or -] 5.3 (condensed milk) (n = 10) Hexane (n = 10) 46.3 [+ or -] 5.7 32.4 [+ or -] 8.3 EtOAc (n = 9) 67.5 [+ or -] 5.8 (a) 16.9 [+ or -] 7.0 MeOH (n = 11) 63.6 [+ or -] 7.5 (a) 20.2 [+ or -] 5.2 All values represent the group mean + s.e.m. (n = 9-11). #o.a. -- number of open arm entries. Time o.a. -- time spent on open arm, #c.a. -- number of closed arm entries, Time c.a. -- time spent on closed arm, #phd -- number of protected head dips, Time phd - time spent on protected head dips, #uphd -- number of un-protected head dips. (a) p < 0.05 vs. control, t-test. Table 4. Comparison of the plus maze performance of rats treated with asiaticoside (1 mg/kg body wt.) and asiaticoside (3 mg/kg body wt.) or the vehicle (peanut oil), 1 h post-treatment Treatment #o.a. Time o.a. (s) #c.a. Time c.a. (s) Control 2.6 [+ or -] 33.4 [+ or -] 15.7 [+ or -] 211.5 [+ or -] peanut oil 0.5 8.5 1.4 7.9 (n = 11) Asiaticoside 4.0 [+ or -] 58.2 [+ or -] 12.0 [+ or -] 160.4 [+ or -] 1 mg/kg 0.8 11.8 1.5 (a) 10.6 (a) body wt. (n = 9) Asiaticoside 4.2 [+ or -] 63.9 [+ or -] 11.1 [+ or -] 168.4 [+ or -] 3 mg/kg 1.3 16.1 0.8 (a) 19.0 (a) body wt. (n = 8) Treatment #phd Time phd (s) #uphd Control 11.2 [+ or -] 1.0 34.6 [+ or -] 3.6 7.4 [+ or -] 2.0 peanut oil (n = 11) Asiaticoside 13.4 [+ or -] 2.3 43.6 [+ or -] 6.8 15.0 [+ or -] 3.3 1 mg/kg body wt. (n = 9) Asiaticoside 11.4 [+ or -] 0.9 36.0 [+ or -] 3.2 20.4 [+ or -] 3 mg/kg 5.3 (a) body wt. (n = 8) All values represent the group mean + s.e.m. (n = 8-11). #o.a. -- number of open arm entries, time o.a. -- time spent on open arm, #c.a. -- number of closed arm entries, time c.a. -- time spent on closed arm, #phd -- number of protected head dips, time phd - time spent on protected head dips, #uphd -- number of un-protected head dips. (a) p < 0.05 vs. control, t-test. Table 5. Comparison of plus maze performance of rats treated with asiaticoside 3 (5 and 10 mg/kg body wt.) with control (peanut oil), after 1 h post-drug interval Treatment #o.a. Time o.a. (s) #c.a. Time c.a. (s) Control 2.5 [+ or -] 32.1 [+ or -] 10.0 [+ or -] 203.4 [+ or -] (n = 11) 0.4 7.0 1.2 12.2 Asiaticoside 4.8 [+ or -] 68.9 [+ or -] 10.3 [+ or -] 144.7 [+ or -] 3 mg/kg 0.5 (a) 8.1 (a) 0.5 7.9 (a) body wt. (n = 12) Asiaticoside 4.8 [+ or -] 64.3 [+ or -] 10.1 [+ or -] 143.6 [+ or -] 5 mg/kg 0.9 (a) 13.8 (a) 0.7 11.9 (a) body wt. (n = 11) Asiaticoside 4.2 [+ or -] 60.5 [+ or -] 10.6 [+ or -] 156.6 [+ or -] 10 mg/kg 0.5 (a) 9.2 (a) 0.7 8.7 (a) body wt. (n = 13) Treatment #phd Time phd (s) #uphd Control 11.0 [+ or -] 47.7 [+ or -] 6.7 13.9 [+ or -] 3.1 (n = 11) 1.5 Asiaticoside 18.4 [+ or -] 66.3 [+ or -] 7.0 25.0 [+ or -] 2.9 3 mg/kg 1.6 (a) body wt. (n = 12) Asiaticoside 18.7 [+ or -] 68.3 [+ or -] 7.4 (a) 23.0 [+ or -] 5.8 5 mg/kg 1.4 (a) body wt. (n = 11) Asiaticoside 16.4 [+ or -] 61.9 [+ or -] 4.8 25.5 [+ or -] 10 mg/kg 1.1 (a) 3.8 (a) body wt. (n = 13) All values represent the group mean + s.e.m. (n = 11-13). #o.a. -- number of open arm entries, time o.a. -- time spent on open arm, #c.a. -- number of closed arm entries, time c.a. -- time spent on closed arm, #phd -- number of protected head dips, time phd - time spent on protected head dips, #uphd -- number of un-protected head dips. (a) p < 0.05 vs. control, t-test. Table 6. Comparison of performance in the open field test of rats treated with asiaticoside (3, 5 and 10 mg/kg body wt.) and the control (peanut oil) after 1 h post-drug interval Treatment #c c.dur Control (n = 10) 7.9 [+ or -] 1.2 24.8 [+ or -] 3.0 Asiaticoside 3 mg/kg 9.2 [+ or -] 0.6 35.9 [+ or -] 3.4 (a) body wt. (n = 10) Asiaticoside 5 mg/kg 9.0 [+ or -] 1.1 41.3 [+ or -] 5.2 (a) body wt. (n = 10) Asiaticoside 10 mg/kg 8.1 [+ or -] 1.6 39.6 [+ or -] 7.8 body wt. (n = 9) Treatment #c.blocks #p Control (n = 10) 20.6 [+ or -] 3.7 8.8 [+ or -] 1.2 Asiaticoside 3 mg/kg 28.2 [+ or -] 3.0 10.1 [+ or -] 0.6 body wt. (n = 10) Asiaticoside 5 mg/kg 27.0 [+ or -] 3.9 9.9 [+ or -] 1.1 body wt. (n = 10) Asiaticoside 10 mg/kg 27.3 [+ or -] 4.8 9.1 [+ or -] 1.6 body wt. (n = 9) Treatment p.dur #p.blocks Control (n = 10) 271.0 [+ or -] 5.1 131.5 [+ or -] 5.5 Asiaticoside 3 mg/kg 261.9 [+ or -] 3.8 130.1 [+ or -] 6.2 body wt. (n = 10) Asiaticoside 5 mg/kg 257.4 [+ or -] 5.1 121.1 [+ or -] 7.1 body wt. (n = 10) Asiaticoside 10 mg/kg 259.5 [+ or -] 7.7 136.0 [+ or -] 12.4 body wt. (n = 9) All values represent the group mean + s.e.m. (n = 9 - 10). #c -- center number of occurrences, c.dur -- center duration (seconds), #c.blocks -- center number of blocks crossed, #p -- perimeter number of occurrences, p.dur -- perimeter duration (s), #p.blocks - perimeter number of blocks crossed (a) p<0.05 vs. control, t-test. Table 7. Comparison of the social interaction test performance of rats treated with asiaticoside (1 and 3 mg/kg body wt.) with control (peanut oil) after 1 h post-drug interval (Test C4) Treatment #SI SI time Control (peanut oil) 34.8 [+ or -] 2.3 206.9 [+ or -] 6.3 (n = 8 in four groups) Asiaticoside 1 mg/kg 29.8 [+ or -] 0.5 209.5 [+ or -] 19.1 body wt. (n = 8 in four groups) Asiaticoside 3 mg/kg 31.8 [+ or -] 2.0 166.6 [+ or -] 16.5 body wt. (n = 8 in four groups) Treatment #Non-int. Non-int. time Control (peanut oil) 35.0 [+ or -] 2.0 207.5 [+ or -] 4.2 (n = 8 in four groups) Asiaticoside 1 mg/kg 29.8 [+ or -] 0.5 (a) 206.0 [+ or -] 19.3 body wt. (n = 8 in four groups) Asiaticoside 3 mg/kg 32.5 [+ or -] 1.9 251.7 [+ or -] 16.6 (a) body wt. (n = 8 in four groups) All values represent the group mean + s.e.m. (n = 4). #SI -- number of social interactions, SI time -- time spent on social interactions, #non-int. -- number of non-interactions, non-int. time -- time spent on non-interactions. (a) p<0.05 vs. control, t-test.