JOURNAL OF MEDICINAL FOODJ Med Food 9 (1) 2006, 113118 Mary Ann Liebert, Inc. and Korean Society of Food Science and Nutrition

Nardostachys jatamansi Improves Learning and Memory in MiceHanumanthachar Joshi1 and Milind Parle2

1Department of Pharmacognosy, SETs College of Pharmacy, Dharwad, Karnataka; and 2Division ofPharmacology, Department of Pharmaceutical Sciences, Guru Jambheshwar University,

Hisar, Haryana, India

ABSTRACT Cure of cognitive disorders such as amnesia, attention deficit, and Alzheimers disease is still far from beingrealized in the field of medicine. Nootropic agents such as piracetam, aniracetam, and choline esterase inhibitors like donepezilare being used for improving memory, mood, and behavior, but the resulting side effects associated with these agents havemade their applicability limited. In Ayurveda, the roots of Nardostachys jatamansi have been clinically employed for theiranti-ischemic, antioxidant, anticonvulsant, and neuroprotective activities. The present study was undertaken to assess the po-tential of N. jatmansi as a memory enhancer. The elevated plus maze and the passive avoidance paradigm were employed toevaluate learning and memory parameters. Three doses (50, 100, and 200 mg/kg, p.o.) of an ethanolic extract of N. jatamansiwere administered for 8 successive days to both young and aged mice. The 200 mg/kg dose of N. jatmansi ethanolic extractsignificantly improved learning and memory in young mice and also reversed the amnesia induced by diazepam (1 mg/kg,i.p.) and scopolamine (0.4 mg/kg, i.p.). Furthermore, it also reversed aging-induced amnesia due to natural aging of mice. Asscopolamine-induced amnesia was reversed, it is possible that the memory improvement may be because of facilitation ofcholinergic transmission in the brain. Hence, N. jatmansi might prove to be a useful memory restorative agent in the treat-ment of dementia seen in elderly persons. The underlying mechanism of action can be attributed to its antioxidant property.

KEY WORDS: amnesia learning memory Nardostachys jatamansi

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INTRODUCTION

ALZHEIMERS DISEASE is a progressive neurodegenerativebrain disorder that occurs gradually and results in mem-ory loss, unusual behavior, personality changes, and ulti-mately death.1 It is the most common form of onset of adultdementia and attention deficit disorders.2 These personalitydifferences interfere with an individuals life, social activi-ties, and relationships. Episodic memory impairment andamnesic syndromes are the key early features of Alzheimersdisease, while semantic memory impairment is a feature offrontotemporal dementia. Amnesic syndrome refers to pro-found loss of the second stage of recent memory due to bi-lateral hippocampal damage.3 Centrally acting antimus-carinic drugs (like scopolamine) impaired learning andmemory of rats4 and humans.5 Benzodiazepine receptor ag-onists such as diazepam and alprazolam have been shownto produce anterograde amnesia in rodents6,7 and humans,8which probably presented the accessory symptoms ofAlzheimers disease. Nootropic agents such as piracetam,9

pramiracetam, aniracetam,10 and choline esterase inhibitorslike donepezil are being primarily used for improving mem-ory, mood, and behavior. However, the resulting chemo-phobia associated with these agents has made their use limited,11,12 and it is worthwhile to explore the utility of tra-ditional medicines in the treatment of various cognitive dis-orders.

According to the Indian system of medicine, Nar-dostachys jatamansi (Family Valerianaceae), commonlyknown as Bhut jata, Nalada, or Spikenard, is a reputed med-hya (intellect-promoting) herb with many medicinal prop-erties, especially on the central nervous system. The rootsare acrid and bitter with a strong aroma, and they are ex-tensively used in Ayurveda to cure tridosha, kapha, lep-rosy, throat trouble, and ulcers.13 The plant is a major com-ponent of karpuradyarka, an Ayurvedic formulation usedfor brain disorders.14

N. jatamansi contains jatamansone and jatamansic acid15and exerts a distinct depressant action on the central ner-vous system, has been found to possess anticonvulsant ac-tivity, and imparts blackness to hair.16 It has been reportedto have an antioxidant effect17 and is very useful in the treat-ment of cerebral ischemia.18

The present study was undertaken to determine the effectof N. jatamansi on memory in mice employing exterocep-tive and interoceptive behavioral models.

Manuscript received 9 September 2004. Revision accepted 1 December 2004.

Address reprint requests to: Dr. Milind Parle, Phamacology Division, Department ofPharmaceutical Sciences, Post Box 38, Guru Jambheshwar University, Hisar-125001,Haryana, India, E-mail: mparle@rediffmail.com

MATERIALS AND METHODSPreparation of extract

The roots of N. jatamansi (Family Valerianaceae) wereprocured from Jadi Buti Sansthan, Gopeshwar, Uttaranchal,India. The plant was authenticated and identified at theBotanical Survey of India, Dehradun. The specimen,HKJ/DN-NJ2, has been kept in the Division of Pharmacol-ogy, Department of Pharmaceutical Sciences, Guru Jamb-heshwar University, Hisar, Haryana, India. The roots weredried in the shade, cleaned, and powdered to prepare an al-coholic extract. One kilogram of moderately powdered rootsof N. jatamansi was extracted by refluxing with 95% ethanolin a Soxhlet extractor for 810 hours. The extract was evap-orated to dryness under reduced pressure and temperatureusing a rotary vacuum evaporator. The yield of dry extractfrom crude powder of N. jatamansi was 13%. The alcoholicextract of N. jatamansi was suspended in a mixture of Tween80/distilled water (DW) in a ratio of 2:8. The suspensionwas orally administered to animals. The volume of admin-istration to mice was 1 mL/100 g of body weight.

DrugsJatamansi powder, diazepam (Ranbaxy, Gurgaon, India),

and piracetam (UCB India Ltd., Gujarat, India) were usedin the present study. Diazepam for injection and piracetamwere diluted in DW and injected intraperitoneally. The vol-ume of injection per mouse was 1 mL/100 g of body weight.

AnimalsSwiss mice of either sex weighing around 18 g (young

mice, 3 months old) and around 25 g (older mice, 7 monthsold) were used in the present study. Animals were obtainedfrom the disease-free animal house of CCS Haryana Agricul-ture University, Hisar. They were acclimatized to the labora-tory conditions for 5 days before behavioral studies. The ani-mals had free access to food and water and were provided withalternate light and dark cycles of 12 hours each. All experi-ments were carried out during daytime from 0900 to 1400hours. The Institutional Animal Ethics Committee approved theexperimental protocol, and care of the animals was performedfollowing the guidelines of the CPCSEA, Department of Ani-mal Welfare, Government of India (Regulation Number 436).

Laboratory modelsInteroceptive behavioral models. Three models were

used: (a) diazepam-induced amnesia, (b) scopolamine-in-duced amnesia, and (c) aging-induced amnesia.

Exteroceptive behavioral models. Elevated plus maze.The elevated plus maze served as the exteroceptive behav-ioral model (wherein the stimulus existed outside the body)to evaluate learning and memory in mice. The procedureand end point applied in the present study for testing learn-

ing and memory were as in the criteria described by inves-tigators working in psychopharmacology and behavioralpharmacology.19 The apparatus consisted of two open arms(16 cm 5 cm) and two covered arms (16 cm 5 cm 12 cm). The arms extended from a central platform (5 cm 5 cm), and the maze was elevated to a height of 25 cm fromthe floor. On the first day, each mouse was placed at the endof the open arm, facing away from the central platform.Transfer latency (TL) was taken as the time taken by themouse to move into one of the covered arm with all four ofits legs. TL was recorded on the first day. If the animal didnot enter into one of the covered arms within 90 seconds, itwas gently pushed into one of the two covered arms, andthe TL was assigned as 90 seconds. The mouse was allowedto explore the maze for 10 seconds and then returned to itshome cage. Memory retention was examined on the secondday, 24 hours after the first days trial.

Passive shock avoidance paradigm. Passive avoidancebehavior based on negative reinforcement was used to ex-amine the long-term memory. The apparatus consisted of abox (27 27 27 cm) having three walls of wood and onewall of plexiglass, featuring a grid floor (3-mm stainlesssteel rods set 8 mm apart), with a wooden platform (10 7 1.7 cm) in the center of the grid floor. The box was il-luminated with a 15-W bulb during the experimental period.Electric shock (20 V AC) was delivered to the grid floor.

Training was carried out in two similar sessions. Eachmouse was gently placed on the wooden platform set in thecenter of the grid floor. When the mouse stepped down andplaced all its paws on the grid floor, shocks were deliveredfor 15 seconds, and the step-down latency (SDL) wasrecorded.20 SDL was defined as the time taken by the mouseto step down from wooden platform to the grid floor withall its paw on the grid floor. Animals showing SDL in therange of 215 seconds during the first test were used for thesecond session and the retention test. The second sessionwas carried out 90 minutes after the first test. When the an-imals stepped down before 60 seconds, electric shocks weredelivered for 15 seconds. During the second test, animalswere removed from the shock-free zone if they did not stepdown for a period of 60 seconds. Retention was tested af-ter 24 hours in a similar manner, except that the electricshocks were not applied to the grid floor. Each mouse wasagain placed on the platform, and the SDL was recorded,with an upper cutoff time of 300 seconds.

Locomotor functionLocomotor activity of control and drug-treated animals

was measured with the help of a photoactometer (INCO,Ambala, India).

Drug protocolThe animals were divided into 31 groups. Each group was

composed of a minimum of five animals.

114 JOSHI AND PARLE

Elevated plus maze. Group I represented the control groupfor young mice (n 6). DW (10 mL/kg, p.o.) was admin-istered for 8 days. TL was noted at 45 minutes after ad-ministration on day 8 and after 24 hours, i.e., on day 9.

Group X served as the control group for aged mice (n 6). DW (10 mL/kg, p.o.) was administered for 8 days. TLwas noted at 45 minutes after administration on day 8 andafter 24 hours, i.e., on day 9.

For Group III, diazepam (1 mg/kg, i.p.) was administeredto young mice, and TL was noted at 45 minutes after in-jection on day 8 and after 24 hours, i.e., on day 9.

For Group IV, scopolamine hydrobromide (0.4 mg/kg,i.p.) was administered to young mice, and TL was noted at45 minutes after injection on day 8 and after 24 hours, i.e.,on day 9.

For Groups II and XIV, piracetam (200 mg/kg, i.p.) wasinjected into young and aged mice, respectively. TL wasnoted at 45 minutes after injection and after 24 hours.

For Groups V and VI, N. jatamansi (50 mg/kg and 100 mg/kg, respectively) was administered orally to youngmice for 8 days. The last dose was given 45 minutes beforesubjecting the animals to the elevated plus maze test. TLwas noted on day 8 and again after 24 hours.

For Groups XI and XII, N. jatamansi (50 mg/kg and 100 mg/kg, respectively) was administered orally to agedmice for 8 days. The last dose was given 45 minutes beforenoting TL on day 8.

For Groups VII and XIII, N. jatamansi (200 mg/kg, p.o.)was administered to young and aged mice, respectively, for8 days. TL was noted on day 8 and again after 24 hours,i.e., on day 9.

For Group VIII, N. jatamansi (200 mg/kg, p.o.) was ad-ministered to young mice for 8 days. At 60 min after ad-ministration of the last dose on day 8, diazepam (1 mg/kg,i.p.) was administered. TL was noted at 45 minutes after ad-ministration of diazepam and after 24 hours, i.e., on day 9.

For Group IX, N. jatamansi (200 mg/kg, p.o.) was ad-ministered to young mice for 8 days. At 45 minutes afteradministration of the last dose on day 8, scopolamine hy-drobromide (0.4 mg/kg, i.p.) was administered. TL wasnoted at 45 minutes after administration of diazepam and af-ter 24 hours, i.e., on day 9.

Passive-avoidance paradigm. Group XV represented thecontrol group for young mice (n 6). DW (1 mL/100 g,p.o.) was administered for 8 days. At 90 minutes after ad-ministration on day 8, SDL was recorded. Retention was ex-amined after 24 hours.

For Groups XVI, XVII, and XVIII, N. jatamansi ethano-lic extract (50, 100, and 200 mg/kg, respectively) was ad-ministered orally for 8 days. SDL was recorded at 90 min-utes after administration on day 8 and after 24 hours.

For Group XIX, scopolamine hydrobromide (0.4 mg/kg,i.p.) was administered to young mice after training on day8, and SDL was recorded at 45 minutes after injection. Re-tention was examined after 24 hours.

For Group XX, N. jatamansi (200 mg/kg, p.o.) was ad-

ministered to young mice for 8 days. At 45 minutes after ad-ministration of the last dose on day 8, scopolamine hydrobro-mide (0.4 mg/kg, i.p.) was administered. SDL was recordedat 90 minutes after administration on day 8 and after 24 hours.

Group XXI represented the control group for aged mice(n 6). DW (1 mL/100 g, p.o.) was administered for 8 days.At 90 minutes after administration on day 8, SDL wasrecorded. Retention was examined after 24 hours.

For Groups XXII, XXIII, and XXIV, N. jatamansi ethano-lic extract (50, 100, and 200 mg/kg, respectively) was ad-ministered orally for 8 days. SDL was recorded at 90 min-utes after administration on day 8 and after 24 hours.

Statistical analysisAll the results were expressed as mean standard error.

The data were analyzed using analysis of variance and Stu-dents (unpaired) t test. A value of P .05 was consideredas statistically significant.

RESULTSWrithing syndrome

The three doses (50, 100, and 200 mg/kg, p.o.) of N. jata-mansi neither produced any sign of writhing syndrome inmice nor elicited any change in the emotional state of ani-mals as reflected by their overall behavioral pattern withinthe home cage.

Effect on locomotor activityIn the present study, N. jatamansi (50, 100, and 200

mg/kg) did not produce any significant change in the loco-motor function of animals (scores of 222.6 8, 218 2,and 211 15, respectively) when tested using a photoac-tometer as compared with the control group (score 216.4 12) when tested using a photoactometer.

Effect on TL using the elevated plus mazeAged mice showed higher TL values on the first day and

on the second day (after 24 hours) as compared with youngmice, indicating impairment in learning and memory (i.e.,aging-induced amnesia). Piracetam (200 mg/kg, i.p.) pre-treatment for 8 days decreased TL on day 8 and after 24hours, i.e., on day 9, as compared with the DW-treatedgroup, indicating improvement in both learning and mem-ory. Scopolamine (0.4 mg/kg) and diazepam (1 mg/kg) in-creased TL significantly (P .05) in young mice on the firstand second days as compared with the control group, indi-cating impairment of memory.

N. jatamansi (50 mg/kg and 100 mg/kg, p.o.) decreasedthe TL on days 8 and 9 in young and aged mice (P .05)when compared with control groups. A higher dose of N.jatamansi (200 mg/kg, p.o.) more significantly enhanced thelearning and memory of aged animals rather than the youngmice as reflected by a marked decrease in TL on days 8 and

N. JATAMANSI IS A POTENTIAL MEMORY ENHANCER 115

9 when the animals were subjected to elevated plus mazetests (Tables 1 and 2). Pretreatment with the higher dose ofN. jatamansi for 8 days successively to young mice pro-tected them (P .05) against scopolamine-, diazepam-, andaging-induced amnesia.

Effect on SDL using the passive avoidance apparatusN. jatmansi extract (200 mg/kg, p.o.) profoundly increased

SDL significantly as compared with the control group on thesecond day, indicating improvement in memory of youngmice. Furthermore, this dose of N. jatamansi reversed scopo-lamine-induced amnesia as well, like in the elevated plus mazemodel (Table 3). Scopolamine hydrobromide (0.4 mg/kg, i.p.)significantly decreased SDL on the second day after training,indicating impairment of memory.

N. jatamansi extract (200 mg/kg, p.o.) administered orallyfor 8 days significantly reversed amnesia induced by scopo-lamine and natural aging (Table 3).

DISCUSSIONAlzheimers disease is a progressive neurodegenerative

disorder characterized by a gradual decline in memory. Nor-mal aging is known to deteriorate memory in human beings.Oxygen free radicals, the harmful by-products of oxidativemetabolism, are known to cause organic damage to the liv-ing system, which may be responsible for the developmentof Alzheimers disease in the elderly.21 In the present study,the older group of animals showed impaired learning andmemory due to the aging process, and N. jatamansi im-proved learning and memory in the aged mice as reflectedby a significant decrease in TL and increase in SDL.

Piracetam, the first representative of a class of nootropicagents, has been shown to improve memory deficits in geri-atric individuals. Repeated injections of piracetam had im-proved learning abilities and memory capacities of labora-tory animals.22,23

The pretreatment with an alcoholic extract of N. jatamansiin cerebral ischemia for 15 days has been shown to produce

116 JOSHI AND PARLE

TABLE 2. EFFECT OF N. JATAMANSI ON TL VALUES OF AGED MICE IN THE ELEVATED PLUS MAZE

TL test

Group Treatment Dose (kg1) Day 8 Day 9

I Control (DW) (young) 10 mL 21.6 30.3 19.34 1.28X Control (DW) (aged) 10 mL 36.97 1.4a 32.11 1.81aXI N. jatamansi 50 mg 24.82 3.13a 18.02 1.59aXII N. jatamansi 100 mg 18.20 3.42b 10.71 1.29bXIII N. jatamansi 200 mg 16.43 5.32b 9.12 1.78bXIV Piracetam 200 mg 18.4 3.1a 12.3 1.9a

Each group consists of five animals each except groups I and X (n 6). Data are mean standard error of the mean values.aP .05 compared with DW control (young).bP .05 compared with DW control (aged).

TABLE 1. EFFECT OF N. JATAMANSI ON TL VALUES OF YOUNG MICE IN THE ELEVATED PLUS MAZE

TL test

Group Treatment Dose (kg1) Day 8 Day 9

I Control (DW) 10 mL 21.63 0.3 19.34 1.28II Piracetam 200 mg 18.04 3.04a 16.46 3.01aIII Diazepam 1 mg 28.02 8.05a 31.66 8.63aIV Scopolamine 0.4 mg 46.4 9.86a 38.61 2.41aV N. jatamansi 50 mg 21.80 2.92 17.95 1.85VI N. jatamansi 100 mg 17.22 1.76a 16.52 0.52aVII N. jatamansi 200 mg 12.34 4.15a 10.13 1.36aVIII N. jatamansi diazepam 100 mg 1 mg 15.3 3.21b 9.64 2.11bIX N. jatamansi scopolamine 100 mg 0.4 mg 17.54 1.81c 11.46 1.04c

Each group consists of five animals each except group I (n 6). Data are mean standard error of the mean values.aP .05 compared with control.bP .05 compared with control (diazepam treated).cP .05 compared with control (scopolamine treated).

a marked improvement in motor coordination and sponta-neous motor activity after 2 hours of occlusion and 22 hoursof reperfusion. N. jatamansi enhanced learning and mem-ory in mice in exteroceptive as well as interoceptive mod-els. Furthermore, N. jatamansi was found to be more potentor equivalent in potency to piracetam in the present inves-tigation, as indicated by the equivalent and small dose, morepronounced efficacy, and shorter time for onset of action.The memory-enhancing activity of N. jatamansi may be at-tributed to the anti-cerebral ischemic action,18 neuroprotec-tive activity,25 and antioxidant properties17 of variousjatanolides present in the herb.

Since N. jatamansi elicited a more pronounced neuro-protective action in aged mice, it may prove to be a usefulmemory-enhancing agent to treat dementia in elderly indi-viduals. Thus N. jatamansi may be of enormous use in de-laying the onset and reducing the severity of symptoms ofAlzheimers disease.

ACKNOWLEDGMENTSThe authors are deeply grateful to Dr. R.P. Bajpai, Hon-

orable Vice-Chancellor, Guru Jambheshwar University,Hisar, for research facilities and motivation. We are alsothankful to UCB India Pvt. Ltd. (Gujarat), for the supply ofpiracetam. We sincerely thank Dr. V.H. Kulkarni, Principal,Sonia Education Trusts College of Pharmacy, Dharwad, forencouragement. We thank Dr. Yoganarasimhan, RegionalResearch Center (Ayurveda), Bangalore, for suggestions andencouragement.

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N. JATAMANSI IS A POTENTIAL MEMORY ENHANCER 117

TABLE 3. EFFECT OF N. JATAMANSI ON SDL VALUES OF USING THE PASSIVE-AVOIDANCE APPARATUS

Group Mice Treatment Dose (kg1) SDL after 24 hours (seconds)

XV Young Control (DW) 10 mL 112 13.2XVI Young N. jatamansi 50 mg 248 16.4aXVII Young N. jatamansi 100 mg 191 2.36aXVIII Young N. jatamansi 200 mg 284 24.62aXIX Young Scopolamine 0.4 mg 16.2 2.19aXX Young N. jatamansi scopolamine 200 mg 0.4 mg 253.6 23.21a,bXXI Aged Control (DW) 10 mL 42.46 6.31XXII Aged N. jatamansi 50 mg 48.18 6.29cXXIII Aged N. jatamansi 100 mg 62.51 4.31cXXIV Aged N. jatamansi 200 mg 98.19 1.96c

Data are mean standard error of the mean values.aP .05 compared with DW control (for young and aged mice).bP .05 compared with scopolamine-treated group alone.cP .05 compared with DW control (aged mice alone).

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