RESEARCH ARTICLE

Neuropsychopharmacology of a therapeutically usedAndrographis paniculata extract: a preclinical study

Ajit Kumar Thakur & Shyam Sunder Chatterjee &

Vikas Kumar

Received: 30 July 2013 /Accepted: 17 October 2013# Institute of Korean Medicine, Kyung Hee University 2013

Abstract Andrographis paniculata (Burm. F.)Wall. Ex Nees(Acanthaceae) also called as Kalmegh , or “King of Bitters”. InAyurveda, Andrographis paniculata is classified as a Rasayanaherb. More than 50 % of the poly-herbal formulations commer-cialized in India for treatment of liver function disorders containAndrographis paniculata . Pilot study and general neurophar-macological screening were conducted with analytically stan-dardized Andrographis paniculata extract (AP), to evaluate itstherapeutic potential for mental health problems. A battery ofrodent behavioral models developed in our laboratory was usedfor characterizing brain function modulating activities. Dailyoral administrations of even very high doses (800 mg/kg) ofthe extract were well tolerated by laboratory rodents without anyapparent behavioral alterations. A single oral dose of the extract(AP 25 to 800 mg/kg) was inactive in mice stress-inducedhyperthermia, and apomorphine induced cage-climbing tests inrats. However, dose and duration of treatment dependent effica-cy of the extract after its daily doses up to 200 mg/kg/day for10 days was observed in both the tests. Moreover, dose depen-dent effects of the extract in suppressing locomotion, potentiat-ing pentobarbital sleep, and antagonizing pentylenetetrazole ormaximal electroshock triggered seizureswere also apparent afterits 10 daily doses up to 200 mg/kg/day. Anxiolytics andantidepressants-like activity of AP treatment was also apparentin rats after 10 daily doses. Therefore, prolonged daily oral

intake of Andrographis paniculata extract gradually suppressescentral sensitivity to acute stressful stimuli, and eventually downregulates central dopaminergic receptors. Benzodiazepines likeanxiolytic and seizure suppressing activities of the extract can beexpected after its daily intake.

Keywords AyurvedicRasayana herb .Adaptogenic activity .

Repeated oral dose . Psychotherapeutic potential

Introduction

Andrographis paniculata (Burm. F.) Wall. Ex Nees (Family:Acanthaceae) also called Kalmegh , or “King of Bitters”, is awildly growing flowering herb, now often cultivated also inmany countries for medicinal purposes (Lattoo et al. 2006).Diverse such uses of the herb have been known in Indiansubcontinent since centuries, and it has been estimated thatmore than 50 % of the multi-ingredient herbal formulationscommercialized in India for treatment of liver function disor-ders contain Andrographis paniculata as an active ingredient(Williamson 2002). Ayurveda, i.e. the oldest traditionallyknown medical system of India, classifies Andrographispaniculata as a Rasayana herb (Govindarajan et al. 2005).Rasayana is one of the eight branches of Ayurvedarecommending diverse combinations of plant derived and othernatural products as nourishing and rejuvenating drugs withmultiple applications for promoting longevity and memoryfunctions. Observations made during more recent decadesadd considerable experimental and clinical evidences justifyingsuch traditionally known medicinal uses of numerousRasayana herbs (Balasubramani et al. 2011). Since all suchherbs studied to-date possess nootropic, cognition enhancing,anti-stress, anxiolytic, antidepressant and other CNS-functionmodulating activities, many of them are now pharmacological-ly classified as herbal adaptogens (Winston andMaimes 2007).

A. K. Thakur :V. Kumar (*)Neuropharmacology Research Laboratory, Department ofPharmaceutics, Indian Institute of Technology (Banaras HinduUniversity), Varanasi 221 005, Indiae-mail: vikas.phe@iitbhu.ac.in

S. S. ChatterjeeStettiner Straße 1, Karlsruhe, Germany

S. S. ChatterjeePharmacology Research Laboratories, Dr. Willmar Schwabe GmbH& Co. KG, Karlsruhe, Germany

Orient Pharm Exp MedDOI 10.1007/s13596-013-0140-4

Although numerous preclinical as well as clinical reportscontinue to reveal broad spectrums of therapeutically interest-ing bio-activities of Andrographis paniculata extracts andtheir constituents, as yet only very little information on itsmental function modulating potentials have appeared (Kumaret al. 2012; Valdiani et al. 2012; Chao and Lin 2010;Jarukamjorn and Nemoto 2008). It has been reported though,that intra-peritoneal administrations of a methanolic extract ofthis plant possess sedative and other brain function alteringactivities in rodent models (Mandal et al. 2001). However,since for therapeutic purposes repeated oral doses ofAndrographis paniculata extracts are used, therapeutic rele-vance of these observations remains uncertain, or question-able. Symptomatic relief obtained from such treatments inpatients suffering from pathologies triggered by systemicinflammation and associated with diverse spectrums mentalhealth problems have been demonstrated by several properlycontrolled clinical trials (Subramanian et al. 2012). Sincealterations in the sensitivity of the brain function are a com-mon characteristic of such pathologies, they are now groupedunder the heading “central sensitivity syndromes” or CSS(Yunus 2008).

Upper respiratory tract infection is one such conditioncommonly associated with diverse spectrums of psychopa-thologies (Smith 2012a, b). Medicinal uses of Andrographispaniculata preparations for symptomatic relief of such con-ditions has been known since long, and a recent report hasreconfirmed such efficacy of an analytically well character-ized Andrographis paniculata extract rich in andrographolide(Saxena et al. 2010). Subsequent efforts to identify the bioac-tive constituents of the extract have indicated that several anti-inflammatory components of the extract other thanandrographolide could also be involved in its observed thera-peutic efficacy (Chandrasekaran et al. 2011, 2010). Sincesystemic inflammation is often associated with mental healthproblems (Dantzer et al. 2008), efforts are now being made inour laboratories to define therapy relevant psychopharmaco-logical activity profile of the extract and to identify its psy-choactive constituents. For such purposes an Ayurveda cen-tered psychopharmacological strategy evolving from our stud-ies with several Rasayana herbs is being used (Chatterjee andKumar 2012). The very first observation made with the ther-apeutically used extract (AP) is described and discussed in thiscommunication.

Material and methods

Animals

Adult Charles Foster albino rats (150±10 g) and Swiss albinomice (20±5 g), of either sex, were obtained from the CentralAnimal House of the Institute of Medical Science, Banaras

Hindu University, Varanasi, India. Prior approval from theCentral Animal Ethical Committee of the University(CAECU) was taken for this study protocol (Dean/11-12/CAEC/325, dated 30-11-2011). The animals were housed ingroups of six in polypropylene cages at an ambient tempera-ture of 25±1 °C and 45–55 % relative humidity, with a12:12 h light/dark cycle. They were always provided withcommercial food pellets and water ad libitum, and wereacclimatized for at least 1 week before using them for theexperiments. Behavioral experiments were conducted be-tween 09.00 and 14.00 h, and “Principles of laboratory animalcare” (NIH publication number 85-23, revised in 1985) guide-lines were always followed.

Plant extract and analytical characterization

The plant material was identified as Andrographis paniculata(Burm. F.) Wall. Ex Nees by in-house botanist at R&D Centerof Natural Remedies Pvt. Ltd., Bangalore, India, and a vouch-er herbarium specimen (No: NR582) is available in theirlaboratories. Details of extraction procedure and analyticalmethod used for standardizing of the tested extract (AP) havebeen reported elsewhere (Chandrasekaran et al. 2009, 2010).Briefly, extraction of Andrographis paniculata coarse groundleaves was done with methanol for 3 h, in a stainless steeljacketed extractor fitted with reflux condenser. The liquidextract was removed and the remaining raw material was re-extracted two more times with methanol in a similar manner.The resulting extracts were combined, concentrated and driedunder vacuum (at <55 °C). The yield of the dried extract was6 % (w/w). After extraction with methanol, the left over rawmaterial was extracted with water for 3 h under reflux condi-tions. This water extract was separated and concentrated undervacuum at around 75 °C followed by spray drying. Two partsof methanolic and one part of successive water extract wereblended to get AP (ratio 2:1). Analytically, the extractc o n t a i n e d a nd r o g r a p ho l i d e ( >3 0 . 0 % , w /w ) ,isoandrographolide (>0.3 %, w/w), neoandrographolide(>1.0 %, w/w), andrograpanin (>0.3 %, w/w), and 14-deoxy-11,12 didehydroandrographolide (<5.0 %, w/w).

Animal grouping and drug treatments

Rats and mice of either sex were equally divided in experi-mental groups of six animals each. Orally administered vol-ume of the extract suspension in 0.3 % carboxymethyl cellu-lose (CMC; Central Drug House, Delhi, India) was 10 ml/kg,and the vehicle control animals were treated accordingly withthe vehicle. In pilot experiments, effects of single and repeateddaily doses of AP 25, 50, 100, 200, 400, 600 and 800 mg/kg/day (p.o.) were evaluated in the same groups of animals ondays 1, 5, 7 or 10 of the experiment. Choice of this dose rangefor this dose finding experiments was based on literature data

A. K. Thakur, et al.

available on dose ranges of diverse other types ofAndrographis paniculata extracts in rodent models. In otherexperiments, AP was always orally administered at dailydoses of 50, 100 and 200 mg/kg/day for ten consecutive days.A standard drug treated group was always run parallelly inthese experiments, and all tests were conducted 60 min afterthe last treatments.

Pilot experiment

In this exploratory experiment the effect of single and repeateddaily doses of the extract were quantified in four well stan-dardized pharmacological models described in the following:

Stress induced hyperthermia test

After 60 min of treatment and initial rectal temperature mea-surement, individual mouse from each group was placed in ablack box (24×29×40 cm) with a grid floor for 1 min. Electricfoot shock through the grid floor (2 mA, 50 Hz of 2 msduration) was delivered for stress induction. After 10 s of theirstay in the box, five consecutive foot shocks of 2 mA at 10 sintervals were given through the grid floor, immediately after

that the animals were placed back in their home cages, andstress induced change in rectal temperature was quantifiedafter 10 min stay in their home cages (Zethof et al. 1994).

Ring test

After 60 min of treatment individual mouse from each groupwas placed on a steel ring (diameter 5.5 cm) fixed to a steelstand at a height of 16 cm. The assay was performed by lifting-up the mouse by its tail and placing the animal gently acrossthe wire ring. During the following 5 min the sum of the timesduring which the mouse remains motionless is noted to thenearest second. The criterion for immobility is the absence ofall voluntary body movements except for those associatedwith breathing. Lack of snout and whisker movements pro-vides an easily recognizable endpoint for the onset of immo-bility and serves to make the test reasonably objective. Thetest was repeated at 90 min and 120 min interval aftertreatment of extract. Immobility was expressed as an ‘im-mobility index’, defined as that percentage of the total timespent on the ring during which the animal remains motion-less (Pertwee 1972).

Immobility index ¼ Immobility period secð Þ.Total time spent on ring secð Þ � 100

Inverted screen test

This test is also known as the wire mesh test or horizontalscreen test. During test, a mouse from each group after 60 minof treatment was put upon the screen (15×15 cm wire mesh)elevated at 40 cm above the floor. The screen is then slowlyrotated by 180° (Coughenor et al. 1977), and grading systemfor the inverted screen test was scored as follow: 0=the mouseclimbs to the top; 1=the mouse fails to reach the top but holdsonto the screen; and 2=the mouse falls from the screen during1 min of the test duration (Maxwell et al. 1993).

Cage-climbing test

This test was done by method described by Protais et al.(1976) in rats with some modifications. Apomorphine(0.50 mg/kg, s.c.; Sigma, St. Louis, MO) was administeredafter 60 min of extract treatment. Immediately after the injec-tion, a rat was put into a cylindrical individual cage, 12 cmdiameter and 14 cm high, with walls of vertical metal bars,2 mm diameter, and 1 cm apart, surmounted by a smoothsurface. After a 5-min-period of exploratory behavior, three

consecutive observations were made on each animal by ablinded observer at 10, 20 and 30 min after apomorphineinjection and these three scores were averaged. This behav-ioral score used was:

0=four paws on the floor, 1=forefeet holding the wall, and2=four paws holding the wall.

General neuropsychopharmacological screening

Experimental procedures described in the following wereused:

Potentiation of pentobarbital induced hypnosis

Pentobarbital (40 mg/kg, i.p.; Loba Chemie Pvt. Ltd., Mum-bai, India) was administered to control and drug treated rats.Onset of sleep (loss of righting reflex), and duration of sleepwas measured by a blind observer (Ojima et al. 1995). The last(10th) doses of AP (50, 100 and 200 mg/kg, p.o.), or of thestandard drug diazepam (5 mg/kg, p.o.) were administered60 min before pentobarbital injection.

Neuropsychopharmacology of a therapeutically used Andrographis paniculata extract

Spontaneous locomotor activity

The spontaneous locomotor activity in photoactometer (Tech-no Electronics, India) was assessed by method described byRamanathan et al. (1999). The last (10th) doses of AP (50, 100and 200 mg/kg, p.o.), or of lorazepam (1 mg/kg, p.o.), wereadministered 60 min before subjecting the animals to sponta-neous locomotor activity test. Each rat was allowed for aperiod of 5 min in a square closed field arena (30×30×30cm) equipped with 6 photocells in the outer wall for sponta-neous locomotion. Number (N) of photocell beams interrup-tion (locomotor activity) was recorded by means of a 6 digitsresettable counter.

Maximal electroshock (MES) seizures in rats

According to this method, the supra-maximal electroshock(150 mA, 50 Hz) was given through a pair of pinnalelectrodes for 0.2 sec duration using a convulsiometer(Techno Electronics, India). The hind limb extensor re-sponse was taken as the positive end point (Kumar et al.2003). Albino rats were pre-screened and only those show-ing positive hind limb tonic extensor response were usedfor the experiment. At least 48 h after pre-screen, drugtreatment was started for 10 consecutive days. The last(10th) doses of AP (50, 100, and 200 mg/kg, p.o.), or ofphenytoin (30 mg/kg, p.o.), were administered 60 min be-fore the MES challenge. The severity of convulsions wasassessed by the duration of tonic flexion, hind limb tonicextensor (HLTE), clonus and stupor phase for each animal(in second).

Pentylenetetrazole (PTZ) induced convulsions in mice

The mice were challenged with PTZ (80 mg/kg, i.p.; Sigma,Bangalore, India). The last (10th) administration of AP (50,100, and 200 mg/kg, p.o.), and of diazepam (10 mg/kg,p.o.) was done 60 min prior to PTZ challenge. The latencyto first myoclonic jerk, percentage clonic seizure, and per-centage protection was recorded by a blind observer(Rudzik et al. 1973).

Anxiolytics-like activity

The two rat models commonly used for assessing benzodiaz-epines like anxiolytic activities of test agents were used. Inthese tests the last (10th) oral daily doses of AP (50, 100, and200 mg/kg), or of lorazepam (1 mg/kg), were administered60 min before subjecting the animals to the two behavioralprocedures summarized below:

Light-dark box (LDB) test

The LDB setup consisted of two compartments; one lit compart-ment (40×50 cm; 350 lux; light box) and one dark compartment(40×30 cm, 70 lux; dark box). The compartments were connect-ed via a small opening (7.5×7.5 cm) enabling transition betweenthe compartments. At the beginning of a 5-min test period eachrat was placed in the centre of the black compartment, facing theopening and (a) number of entries into the white compartment(defined as placing both forepaws into the white compartment),(b) time spent in the white compartment and (c) latency until thefirst entry into the white compartment were assessed by blindedobserver (Henniger et al. 2000; Jurek et al. 2012).

Elevated plus maze test

The method of Pellow and File (1986) was followed. Theelevated plus-maze consisted of two opposite arms, 50×10 cm, crossed with two enclosed arms of the same dimensionbut having 40 cm high walls. The arms were connected with acentral square, 10×10 cm, giving the apparatus shape of a plussign. The maze was kept in a dimly lit room and elevated50 cm above the floor. Rat was placed individually in centre ofthe maze, facing an enclosed arm. Thereafter, number ofentries (all four paws of the rat were in that arm) and timespent on the open and enclosed arms were recorded during thenext 5 min by a blinded observer.

Antidepressants-like activity

The last (10th) doses of AP (50, 100 and 200mg/kg, p.o.), or ofimipramine (15 mg/kg, p.o.), were administered 60 min beforesubjecting the animals in behavior test described below:

Forced swimming test

The method of Willner (1984) was followed. In short, a ratwas individually placed in a cylinder (45×20 cm) containing38 cm water (25±2 °C), so that it could not touch the bottomof the cylinder with its hind limb or tail, or climb over the edgeof the chamber. Two swim sessions were conducted, an initial15 min pre-test on day 9 of drugs treatment, followed by a5 min test, 24 hr later (on day 10). The period of immobility(remained floating in water without struggling and makingonly those movements necessary to keep its head above water)during 5 min test period was recorded.

Statistical analysis

Mean ± standard error of mean (SEM) were calculated for theobserved values in each experimental group. Statistical anal-ysis was performed by one way analysis of variance(ANOVA) followed by Student-Newman-Keuls multiple

A. K. Thakur, et al.

comparison test. GraphPad Prism-5 software was used forstatistical analysis. A p-value less than 0.05 were consideredas statistically significant.

Results

Pilot study

Stress induced hyperthermia

No statistically significant effects of AP treatments on footshock stress triggered transient hyperthermia was detected

after its acute oral doses (results not shown), and no effectsof the extract on basal core temperatures of unstressed animals(before foot stress) were observed after its single oral doses. Itis apparent from the results summarized in Fig. 1 that the basalcore temperature of the vehicle treated control group increasedgradually on the subsequent days of the experiment. Suchdaily handling induced hyperthermia were not observed inany of the AP treated groups, the mean values of which inthese groups on days 1, 5, 7 and 10 were not significantlydifferent from each other. Despite significant (p <0.05) in-creases in basal core temperature of the control group on days5, 7 and 10 of the experiment, the observed foot shocktriggered hyperthermia in this group remained constant inmagnitude on all these test days (Fig. 2a). On the 5th day ofthe treatments, significant antagonistic effect of AP againststress induced hyperthermia was observed after its 100 and200 mg/kg/day doses only. Continuation the treatments for7 days increased somewhat its efficacy, and significant treat-ment effects on this day were observed after its doses between100 to 600 mg/kg/day. However, on this day, the efficacies of400 and 600mg/kg doses of APwere lower than that observedafter its 200 mg/kg daily doses. On day 10 of the treatmentssignificant (p <0.05) antagonistic effects of AP was observedafter all its tested doses higher than 100 mg/kg (Fig. 2a). Thedose-effect curves obtained from these values are shown inFig. 2b, c and d.

Fig. 1 Effects of different dose (a) on basal core temperatures of mice.n =6/group, *=p <0.05 vs. control

Fig. 2 a Effect of repeated treatment of Andrographis paniculata (AP)on stress induced hyperthermia in mice. Log dose response curves ob-tained from the data summarized in this figure are shown in (b), (c) and

(d). n =6/group, *=p <0.05 vs. control. Dotted line represents the pointsof respective temperature values and continuous line represents a curve-fit (ED50 shift) line for log-dose response

Neuropsychopharmacology of a therapeutically used Andrographis paniculata extract

Catalepsy and muscle relaxation

In the ring test, no effects of AP were observed after its singleor 5 or 7 or 10 daily doses tested (results not shown). Simi-larly, no effects of AP treatments were detected in the invertedscreen test for detecting muscle relaxing, or for assessingpotential neurotoxicity of test agents (results not shown).During these tests no effects of AP on body weights or onbehavior of animals in their home cages were observed. Theseobservations reaffirm that oral doses of AP up to 800 mg/kgadministered daily for ten consecutive days are well toleratedby the animals, and do not cause any extra pyramidal sideeffects.

Cage-climbing test

Many psychoactive gents stimulating central dopaminergicsystem induce stereotypic and other behavioral changes inrodents. Apomorphine is one such clinically used dopamineagonist commonly used to test the involvement of centraldopaminergic system in the modes of actions of anxiolytic,sedative, or antipsychotic agents. The cage-climbing test usedin this study is a convenient one for quantifying such effects.A single or 5 or 7 daily oral dose of AP up to 800 mg/kg doesnot influence apomorphine induced cage-climbing behavior inrats (result not shown). However, statistically significant anti-

dopaminergic effects of the extract were observed after its 10daily 100 mg/kg or higher oral doses. The results summarizedin Fig. 3a, and the corresponding dose response curve shownin Fig. 3b, clearly reveal that the maximal possible anti-dopaminergic effect of AP in this test is achieved after its 10daily 200 mg/kg oral doses.

General neuropsychopharmacological screening

Potentiation of pentobarbital-induced hypnosis

Results summarized in Fig. 4 reveal that daily treatment withAP for 10 consecutive days shortens sleep induction periodand prolongs duration of sleep induced by pentobarbital.These statistically significant (p <0.05) effects of the extractwere observed after its two highest doses tested (i.e. 100 and200 mg/kg/day), and qualitatively the efficacy of 200 mg/kg/kg dose of AP was almost equal to those of the anxiolyticdiazepam (5 mg/kg).

Spontaneous locomotor activity

Significant dose dependent inhibitory effect of 10 daily oral100 and 200 mg/kg doses of AP on locomotor activity countswere observed (p <0.05) . Qualitatively though, the observed

Fig. 3 a Effect of 10 daily oral doses of AP on apomorphine (Apo)induced rats and (b) Log dose response curve for 10 daily oral doses ofAP on apomorphine (Apo) induced rats in cage climbing test. n =6/group,

*=p <0.05 vs. control (CMC); ¥=p <0.05 vs. control (Apo). Dotted linerepresents the points of respective temperature values and continuous linerepresents a curve-fit (ED50 shift) line for log-dose response

Fig. 4 Effect of 10 daily oral doses of AP on (a) onset of sleep, and (b) duration of sleep induced by pentobarbitone sodium (40mg/kg, i.p.) in rats. n =6/group, *=p <0.05 vs. control

A. K. Thakur, et al.

effects of the highest AP dose tested was lower than that of1 mg/kg dose of the standard anxiolytic lorazepam (Fig. 5).

Maximal electroshock seizures in rats

Ten daily oral 50 and 100 mg/kg doses of AP had no signif-icant anticonvulsant activity in various phases of MES in-duced seizures like tonic flexion, hind limb tonic extension(HLTE), clonus and stupor. However, the highest AP dosetested (200 mg/kg/day, p.o.) did show significant (p <0.05)anticonvulsants like efficacy in this test. Efficacy of the stan-dard anticonvulsant drug phenytoin (30 mg/kg, p.o.) wasmuch higher than that of the extract (Table 1).

Pentylenetetrazole induced convulsions in mice

Oral daily doses of 50 and 100 mg/kg AP for 10 days had nosignificant anticonvulsant activity quantified as latency inmyoclonic jerk and percentage clonic seizure by PTZ chal-lenged mice. However, significant (p <0.05) anticonvulsantactivity of the highest daily AP dose tested (200 mg/kg/day)and that of the standard anxiolytic diazepam (10 mg/kg, p.o.)were apparent in this test (Table 2).

Anxiolytic activity

Light-dark box (LDB) test

As summarized in Fig. 6a, mean latencies of the all AP treatedgroups (50, 100 and 200 mg/kg/day for 10 consecutive days)

to enter the light chamber of the test box were significantly(p <0.05) lower than that of the vehicle treated control group.The mean numbers of entries (Fig. 6b) and duration of resi-dency time (Fig. 6c) of the all AP treated groups were alsosignificantly (p <0.05) higher than the control group. Qualita-tively the efficacy of 200 mg/kg dose of AP for all the threeparameters quantified were similar to those observed for 1 mg/kg daily oral doses of the standard anxiolytic lorazepam.

Elevated plus maze test

Results of this test are summarized in Fig. 7. Statisticallysignificant (p <0.05) increases in mean numbers of entries(Fig. 7a) and mean time spent in the open arm (Fig. 7b) ofthe all the AP (50, 100, and 200 mg/kg), or lorazepam(1 mg/kg), groups were observed. These observations recon-firm that ten daily 50mg/kg oral doses of AP is high enough toobserve its significant anxiolytic like activities in rat models.

Antidepressant activity

Forced swimming test

Effects of AP and imipramine on the duration of immobilityduring force swimming test are shown in Fig. 8. The extractsat doses of 50, 100 and 200 mg/kg significantly (p <0.05)decreased the duration of immobility (sec) in a dose-dependent manner compared to vehicle treated control rats.Qualitatively, the effect of AP at dose of 200 mg/kg wasanalogous to that of standard drug (imipramine 15 mg/kg)treated group.

Discussion

Adaptogens modulate physiological responses to physical,environmental and emotional stress, and thus help to regulateand support the interconnected functions of neuroendocrineand immune system. Transient hyperthermia triggered bydiverse stressful stimuli is a physiological response, the inten-sity of which is reduced by benzodiazepines therapeutically

Fig. 5 Effect of 10 daily oral doses of AP on spontaneous locomotoractivity in rats. n =6/group, *=p <0.05 vs. control

Table 1 Effect of 10 daily oraldoses of AP on maximal electro-shock (MES) induced seizures inrats

n =6/group, *=p <0.05 vs.control

Treatmentgroups

Time (sec) in various phases of convulsion

Tonic flexion Hind limb tonicextension (HLTE)

Clonus Stupor

Control (0.3 % CMC) 3.83±0.31 13.67±0.42 12.67±0.88 163.17±3.89

AP (50 mg/kg) 3.33±0.49 13.17±0.79 11.83±1.08 160.33±4.45

AP (100 mg/kg) 3.17±0.31 12.33±0.99 10.67±0.61 154.33±3.15

AP (200 mg/kg) 2.50±0.22* 10.33±0.95* 9.50±0.56* 147.67±3.38*

Phenytoin (30 mg/kg) 2.17±0.17* 6.50±0.43* 5.50±0.50* 101.17±3.70*

Neuropsychopharmacology of a therapeutically used Andrographis paniculata extract

used for treatments exaggerated anticipatory anxiety.Although many Rasayana herbs also possess such efficacyin animal models, unlike conventionally known anxiolyticsthey do not induce hypothermia, muscle relaxation and otherneurological deficits even after their higher doses. Results ofthis reported pilot experiments reveal that even the highestdaily oral dose of AP tested (800 mg/kg) was well tolerated byexperimental animals and did not induce muscle relaxationand other adverse effects quantified in unstressed animals.However, clear dose and duration of treatments dependentsuppressive effects of AP on stress induced hyperthermia

was apparent after its repeated daily doses. Quantitativelythough, efficacy of the extract observed on the days 5 and 7of the treatments after its highest two doses (400 and 800 mg/kg/day) were lower than those of its lower tested ones. How-ever, such was not the case on day 10 of the treatments whenefficacies of all doses of AP higher than 200 mg/kg/day werealmost equal (Fig. 2).

Another interesting observation made during the stressinduced hyperthermia test was that the daily handling inducedincreases in basal rectal temperature observed in the controlgroup were complete absent in all AP treated groups (Fig. 1).Since analogous observations were often made in our labora-tories with a few other adaptogenic plant extracts this seems tobe a common characteristic of adaptogens. It must be notedthat maximal possible effects of AP against daily handlinginduced increases in basal rectal temperatures were observedafter its lowest dose tested, which was not the cases for footshock stress triggered transient hyperthermia. These observa-tions could indicate that anti-stress efficacy of AP in regulat-ing or maintaining body temperature depends on the stressintensity felt by the animals.

Body temperature regulation is a fundamental homeostaticfunction that is governed by central nervous system in animalsand human, and abnormalities in the central dopaminergic

Table 2 Effect of 10 daily oral doses of AP on pentylenetetrazoleinduced convulsions in mice

Treatment groups Myoclonic jerklatency (sec)

Clonic seizure(%)

Protection(%)

Control (0.3 % CMC) 54.17±3.41 100 0

AP (50 mg/kg) 59.67±3.42 100 0

AP (100 mg/kg) 62.17±3.70 100 0

AP (200 mg/kg) 66.83±3.28* 83 33

Diazepam (10 mg/kg) 75.00±4.44* 50 83

n =6/group, *=p <0.05 vs. control

Fig. 6 Effect of 10 daily oral doses of AP on rats during light-dark box test (a) latency to entry, (b) number of entry and (c) time spent in light box. n =6/group, *=p <0.05 vs. control

A. K. Thakur, et al.

system causes malignant hyperthermia (Nakamura 2011;Ahuja and Cole 2009). Dose dependent antagonistic effectsof AP observed after its ten daily oral doses in the apomor-phine induced cage-climbing test (Fig. 3) could indicate theinvolvement of central dopaminergic system in the observedanti-hyperthermic effects. It must be noted though that unlikein the stress induced hyperthermia test no significant effects ofthe extract were observed after in the cage-climbing test on the5th and 7th treatment days. The possibility that this discrep-ancy is due to the uses of different rodent species cannot beruled out. It remains certain though, that repeated daily treat-ments with AP is necessary for observing its brain functionmodulating activities, and that AP treatments can ultimatelydepress the sensitivity of the central nervous system againststress and dopaminergic stimuli.

Stress induced hyperthermia tests are sensitive and specificones for identifying benzodiazepines like anxiolytics, withsedative and anti-epileptic activities. Observations that 10daily oral doses of AP also significantly reduce spontaneous

motility and potentiate pentobarbital induced sleep indicate itsbenzodiazepines like sedative effects after its 100 or 200 mg/kg daily oral doses. However, in both the seizure models usedsignificant protective effects of such treatments were observedonly after its highest dose tested, and its efficacy was muchlower than those of the standard antiepileptic drugs testedsimultaneously. These observations could indicate thatprolonged treatments with AP could be useful for helpingpatients with exaggerated anxiety, and other stress triggeredmental health problems commonly associated with numerouschronic diseases. That such could indeed be the case is alsoindicated by the observations made in the behavioral tests fortherapeutically used anxiolytics and antidepressants (Fig. 6, 7and 8). It was interesting to note that statistically significantanxiolytics and antidepressant like effects of AP were ob-served even after its lowest dose tested (50 mg/kg), whereassignificant effects of the extracts in all other tests used in thisstudy were observed only after is 100 mg/kg, or higher doses.Only other statistically significant effects of AP observed after50 mg/kg daily doses during our efforts was its ability tocompletely block daily handling triggered elevation in thebasal core temperature (see Fig. 1). These observations couldindicate that alterations in the sensitivity of thermoregulatorymechanisms and processes could be involved in the therapeu-tically interesting neuro-psychopharmacological activity pro-file of the tested extract. However, more detailed dose re-sponse and other studies are necessary for experimentallyverifying this possibility.

In any case, the observations reported in this article addfurther experimental evidence to our conviction that clinicallyobserved therapeutic benefits of Andrographis paniculataextracts could as well be due to their modulating effects oncentral sensitivity to external stimuli, and that the stress in-duced hyperthermia test can be a useful starting point for

Fig. 7 Effect of 10 daily oral doses of AP on rats in elevated plus maze test (a) number of entry and (b) time spent in open arms. n=6/group, *=p <0.05vs. control

Fig. 8 Effect of 10 daily oral doses of AP on rats in behavior despair test.n =6/group, *=p <0.05 vs. control

Neuropsychopharmacology of a therapeutically used Andrographis paniculata extract

identifying adaptogenic potentials of test agents. Based on theobservations reports in this article, efforts are now being madein our laboratories to identify bioactive constituents of APinvolved in its observed anti-stress activity and to gain moreprecise information on its psychopharmacological activityprofile and modes of actions.

Conclusion

The reported observations strongly suggest that like other wellknown adaptogenic herbs, Andrographis paniculata extractscould as well be a therapeutic option for combating mentalhealth problems commonly associated with diverse non-communicable diseases.

Acknowledgments Ajit Kumar Thakur graciously acknowledged theDepartment of Science and Technology, Govt. of India, New Delhi forawarding INSPIRE Fellowship (IF110595). Authors would like to ac-knowledge Natural Remedies Pvt. Ltd., Bangalore for providing comple-mentary sample of standardized extract of Andrographis paniculata .

Conflicts of interest The authors declare that they have no conflicts ofinterest.

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