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Evaluation of anxiolytic properties of Gotukola--(Centella asiatica) extracts and asiaticoside in rat behavioral models.

Abstract

The ayurvedic medicinal plant Gotukola (Centella asiatica) was evaluated for its anxiolytic properties. Specifically, this study assessed the effects of: Gotukola plant materials of different genotypic origin; hexane, ethyl acetate 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), open field, social interaction, locomotor activity, 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 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

Introduction

Gotukola (Centella asiatica L. Apiaceae) is a psychoactive medicinal plant that has been used for centuries in Ayurvedic medicine 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 in healthy humans. This effect was most pronounced 60min after treatment. In animals, Gotukola increases pentobarbitone-induced sleeping time and decreases immobility 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 of Gotukola was reported to have cognitive-enhancing as well as antioxidant effects in rats (Kumar and Gupta, 2002).

The most prominent group of biologically active compounds isolated from Gotukola is the terpenes (Shukla et al., 1999a). Asiaticoside is the most abundant triterpene glycoside, which is effective in wound healing 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 asiatic acid in vivo by hydrolysis. 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 associated with the dementia of Alzheimer's disease.

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 animals

Test protocols were reviewed and approved by the University of Ottawa Animal Care Committee, according to guidelines of the Canadian Council on Animal Care. Male Sprague-Dawley (SD) rats (300-325 g body wt., each) raised in a pathogen-free colony, were obtained from Charles River 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 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 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) 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.

Locomotor activity

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 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

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.

Drug preparation

Ayurveda recommends mixing Gotukola with milk before its administration in order to achieve the intended psychoactive effects (Sushruta, ~600BC). Dry powder and fresh leaf extracts of Gotukola were sonicated for 15min in 50% sweetened condensed milk solution (Sunfresh Ltd., Toronto, Ontario) or distilled water, to make suspensions. The treatments were given orally (p.o.) using a gavage tube (gage 18, length 5 cm). Asiaticoside was sonicated in peanut oil 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) containing relatively higher or lower concentrations of asiaticoside were selected for this study. The first product was dried leaf material grown from a genotype 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. High Performance Liquid Chromatography (HPLC) analysis of asiaticoside and asiatic acid was undertaken using a validated method reported elsewhere (Wijeweera, 2003). Separations were achieved using a YMS 3-[micro]m ODS 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 in 8min, 80 100 MeCN for 2min, hold 2.5 min, 100-10% MeCN for 2.5 min. Plant material (1 g) was defatted 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 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.

Statistical analysis

The main focus of the study was to investigate the anxiolytic activity of different phytochemical components of Gotukola, compared to the controls. Based on the prior assumptions, post hoc 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 tail in asiaticoside. The more lipophilic 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.

Conclusions

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 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.

Acknowledgments

This study was funded by grants from the International Development Research Centre and the Natural Sciences and Engineering Research Council of Canada.

References

Bradwejn, J., Zhou, Y., Koszycki, D., Shlik, J., 2000. A double blind, placebo-controlled study on the effects of Gotu Kola-(Centella asiatica) on acoustic startle response in healthy subjects. J. Clin. Pharmacol. 20, 680-684.

Diwan, P.V., Karwande, I., Singh, A.K., 1991. Anti-anxiety profile of Manduk Parni (Centella asiatica) in animals. Fitoterapia VXII (3), 253-257.

Inhee, M.J., Eun, S.J., Hwan, Y.S., Kyoon, H., Young, K.J., Keun, P.H., Sang-Sup, J., Whan, J.M., 1999. Protective effects of asiaticoside derivatives against beta-amyloid neurotoxicity. J. Neurosci. Res. 58 (3), 417-425.

Kumar, M.H.V., Gupta, Y.K., 2002. Effect of different extracts of Centella asiatica on cognition and markers of oxidative stress in rats. J. Ethnopharmacol. 79 (2), 253-260.

Lucia, R.D., Sertie, J.A.A., Camargo, E.A., Panizza, S., 1997. Pharmacological and toxicological studies on Centella asiatica. Fitoterapia LXVIII (5), 413-416.

Merali, Z., Levac, C., Anison, H., 2003. Validation of a simple ethologically relevant paradigm for assessing anxiety in mice. Biol. Psychiat. 54, 552-565.

Sakina, M.R., Dandiya, P.C., 1990. A psycho-neuropharma-cological profile of Centella asiatica. Fitoterapia 45 (4), 291-296.

Sang-Sup, J., Chi-Hyoung, Y., Doo-Yeon, L., Heeman, K., Inhee, M.J., Min, W.J., Heesung, C., Young-Hoon, J., Heedoo. K., Hyeung-Geun, P., 2000. Structure-activity relationship study of asiatic acid derivatives against beta amyloid (A-[beta])-induced neurotoxicity. Bioorg. Med. Chem. Lett. 10, 119-121.

Shukla, A., Rasik, A.M., Dhawan, B.N., 1999a. Asiaticoside-induced elevation of antioxidant levels in healing wounds. Phytother. Res. 13 (1), 50-54.

Shukla, A., Rasik, A.M., Jain, G.K., Shankar, R., Kulsh-restha, D.K., Dhawan, B.N., 1999b. In vitro and in vivo wound healing activity of asiaticoside isolated from Centella asiatica. J. Ethnopharmacol. 65 (1), 1-11.

Sushruta, 1991. ~600 BC. Sushruta Samhita, 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, 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, Health Canada, Ottawa, Canada

*Corresponding author. Tel.: + 1 613 562 5262; fax: + 1 613 562 5765.

E-mail address: jarnason@science.uottawa.ca (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.
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Author:Wijeweera, P.; Arnason, J.T.; Koszycki, D.; Merali, Z.
Publication:Phytomedicine: International Journal of Phytotherapy & Phytopharmacology
Article Type:Clinical report
Geographic Code:1CANA
Date:Nov 1, 2006
Words:6715
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