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Planta MedicaJournal of Medicinal Plant and Natural Product Research

www.thieme.de/fz/plantamedica l www.thieme-connect.com/ejournals

bAbstract!

The aim of the present study was to investigatethe antistress potential of Labisia pumila aqueousextract (LPPM/A003) using a battery of testswidely employed in different stressful situations.Pretreatment of experimental animals withLPPM/A003 caused an increase in the swimmingendurance and hypoxia time and also showedthe recovery of physical stress-induced depletionof neuromuscular coordination and scopolamineinduced memory deficit. LPPM/A003 at gradeddoses reversed the chronic restraint stress (RST),induced depletion of CD4+ and CD8+ T lympho-cytes, NK cell population, and corresponding cy-tokines expression besides downregulating the

stress-induced increase in plasma corticosterone,a major stress hormone. In addition, LPPM/A003reversed the chronic stress-induced increase inadrenal gland weight, serum alanine aminotrans-ferase (ALT), alkaline phosphatase (ALP), and he-patic lipid peroxidation (LP) levels and aug-mented the RST induced decrease in hepatic glu-tathione (GSH), thymus and spleen weight. Thus,we conclude that LPPM/A003 has the ability to re-verse the alterations produced by various stress-ful stimuli and therefore restores homeostasis.

Supporting information available online athttp://www.thieme-connect.de/ejournals/toc/plantamedica

Protective Effect of Labisia pumila on Stress-Induced Behavioral, Biochemical, and ImmunologicalAlterations

Authors Kiranjeet Kour1, Neelam Sharma1, Bal Krishan Chandan1, Surrinder Koul2, Payare Lal Sangwan2, Sarang Bani1

Affiliations 1 Department of Pharmacology, Indian Institute of Integrative Medicine, Jammu Tawi, India2 Natural Product Chemistry Division, Indian Institute of Integrative Medicine, Jammu Tawi, India

Key wordsl" Labisia pumilal" Myrsinaceael" stressl" cytokinesl" corticosterone

received October 19, 2009revised January 28, 2010accepted February 7, 2010

BibliographyDOI http://dx.doi.org/10.1055/s-0029-1240953Published online March 9, 2010Planta Med 2010; 76: 1–9© Georg Thieme Verlag KGStuttgart · New York ·ISSN 0032‑0943

CorrespondenceDr. Sarang BaniDepartment of PharmacologyIndian Institute of IntegrativeMedicineCanal RoadJammu TawiJammu and Kashmir – 180001IndiaPhone: + [email protected]

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Introduction!

In the recent years, stress has become an impor-tant aspect of the daily life of every individual thatmay be elicited by environmental, social, or path-ological conditions resulting into changes in thenervous, endocrine, and immune systems. Con-siderable evidence published in the last decadehas focused on alterations at neurochemical, bio-chemical and molecular levels caused by stress inthese systems [1–5]. Stress heightens the risk foradverse health outcomes by suppressing the im-mune response in a fashion that leaves the hostvulnerable to opportunistic infections. Some ofthe common symptoms of stress include depres-sion, anxiety, fatigue, sleep problems and others.An adaptogen/antistress agent is an herbal sub-stance that helps adjust the bodyʼs response onthe cellular level to various stresses put upon it.This results in a regulating effect on many of thebodyʼs systems including immune, nervous, en-docrine system, etc. More specifically, it helps tobring about homeostasis and helps the body todeal more effectively with stress. The use of me-

dicinal plants as a source of relief and cure fromvarious illnesses is as old as humankind itself.Since the introduction of antistress agents/adap-togens, several plants have been investigated,which were once used as tonics in traditionalmedicine due to their adaptogenic and rejuvenat-ing properties [6]. Labisia pumila or Kacip Fati-mah or Akar Fatimah [7], as it is popularly knownin Malaysia, is a plant of very high reputation inthe Malaysian traditional system of medicine. Ac-cording to use, the water decoction of the root orthe whole plant is consumed by Malaya womenfor induction and facilitation of labor [8]. It hasbeen claimed to be used as an anti-dysmenorrhea[9] help for cleansing and avoiding painful or dif-ficult menstruation. The plant also is believed tohelp firm and tone the abdominal muscles. Takinginto consideration the multipurpose use of thisplant in Malaysia, the present study was under-taken to evaluate the normalizing effect of L. pum-ila aqueous extract (LPPM/A003) on alterationsinduced by various types of stressful situationsusing well-established models of stress in ani-mals.

Kour K et al. Protective Effect of… Planta Med 2010; 76: 1–9

Fig. 1 HPLC of Labisia pumila (LPPM/A003).

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Materials and Methods!

Plant materialPlant material was collected from Kawasan Tasik Banding, Grik,Perak, Malaysia on November 2007 and authenticated by the For-est Research Institute of Malaysia. A voucher sample (accessionno. 21648) is retained and deposited at the herbarium of this in-stitute.

Preparation of LPPM/A003 extractDry powdered leaves of L. pumila (500 g) were soaked in distilledwater (4.0 L), and the contents were stirred at 70°C for 3 hrs. Thecontents were then filtered, the marc was taken in 4.0 L of freshwater, and the process was repeated twice (total extractions =three) keeping the temperature and time parameters constant.The total filtrate (~ 10 L) was concentrated at 50°C, reduced to 2-L volume, and then freeze dried into 47.5 g light brownish freeflowing powder labeled as LPPM/A003. The drug-extract ratio(DER) was 100:9.5 (w/w).

HPLC of LPPM/A003The HPLC analysis was done on a Bruker Daltonics Esquire 3000(LC) Agilent 1100 series instrument using RP-18 (Merck; 5 µm,4 × 250mm) column, temp 30°C at wavelength 270 nm with aflow rate of 0.5mL/min. Gradient elution was used with solventA (water containing 0.1% formic acid) and solvent B (acetonitrile)as the mobile phase:

Time (mins): 0 5 50 55 60 65Solvent B (%): 5 5 55 55 5 5

The aqueous extract was standardized on the basis of markercompounds gallic acid and pyrogallol, isolated and well charac-terized from the leaf extract of the plant. On quantification, theconcentration of gallic acid and pyrogallol was found to be1.98% and 0.0015%, respectively. However, the extract was foundto be rich in polyphenolic compounds. HPLC of LPPM/A003 isshown in l" Fig. 1. The HPLC graph of standard gallic acid (Rt =4.60min) and pyrogallol (Rt = 6.07min) at wavelength 270 nmand 254 nm (Fig. 2S), the calibration curve for gallic acid and py-rogallol (LPM-2) (Fig. 3S), and the HPLC graph of LPPM/A003withseparation of gallic acid (Rt = 4.64min) and pyrogallol (Rt = 6.31)at two different wavelengths (Fig. 4S) are presented as Support-ing Information.

ChemicalsFluorescein isothiocyanate (FITC)-labeled CD4 anti-mousemono-clonal antibody, phycoerytherin (PE)-labeled CD8 anti-mousemonoclonal antibody, PE labeled CD244 anti-mouse monoclonalantibody, PE labeled interferon gamma (IFN-γ) anti-mousemonoclonal antibody, PE labeled interleukin-4 (IL-4) anti-mousemonoclonal antibody, PE labeled interleukin-2 (IL-2) anti-mousemonoclonal antibody (BD Biosciences), FACS lysing solution (BDBiosciences), FACS permeabilizing solution, Golgi plug (proteintransport inhibitor; BD Biosciences), phosphate buffer saline(PBS; Sigma Aldrich), and corticosterone Elisa kit (Neogen Corpo-ration) were used for the study.

AnimalsMale Swiss albino mice weighing about 20–22 g and male Wistarrats weighing about 180–200 g were employed for the study. Allthe animals were obtained from the animal house of our institute


(IIIM, Jammu). They were housed in isolators at an ambient tem-perature of 25 ± 2°C and 45–55% relative humidity, with a 12-hlight/dark cycle. The animals used for experimental work wereduly approved by our Institutional Animals Ethics Committee(IAEC) after verifying the protocols that were followed for carry-ing out the experiments. Recommended guidelines for the careand use of animals were strictly followed “Guide for care anduse of laboratory animals” (NIH Publication No. 85–23, revised1985).

Administration of the drugTest material was prepared in distilled water and administereddaily for the duration of the experiment at the dose levels of 25,50, 100, and 200mg/kg per oral (p.o) in all the experiments. Ko-rean Ginseng (KG), a well-known adaptogen was used as a posi-tive control at the dose level of 100mg/kg. A standardized com-mercial sample of KG, containing 20.63% ginsenosides was pur-chased from Shanghai Honghao Chemical Co., Ltd. (Batch. No.20071008).

Acute safety studiesThe acute oral safety studies were carried out following OECD[10] guidelines No. 423 in mice. The animals were observed indi-vidually after dosing at least once during the first 30min, periodi-cally during the first 24 h, and daily thereafter, for a total of 14days, for mortality or any change in general behavior.

Experimental protocolsSwimming endurance test: Male Swiss albino mice were dividedinto six groups (n = 6); one group served as a normal control andthe animals treated with KG (100mg/kg) served as the standardgroup. Test material was administered at the dose level of 25, 50,100, and 200mg/kg, p.o. once daily for 14 days. On day 15, onehour after drug administration, the swimming time of each ani-mal was measured individually. The animals were allowed toswim inside a perplex glass beaker (30 cm high with 20 cm diam-eter, containing water up to 25 cm height maintained at 26 ± 1°C)[11,12]. The mice were allowed to swim, and the moment theygot exhausted and started drowning was considered as the end-point.Antifatigue effect:Antifatigue effect was determined by themeth-od given by Dua et al. [13]. The percent effect of each group wascalculated on the basis of the number of mice that stayed on the

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rota-rod for > 180 seconds (all-or-non response). The same ani-mals were again placed on the rota-rod after 30 minutes of re-moval from the swimming bath, to monitor the antifatigue effectonce again.Hypoxia test: Drug administration was carried out continuouslyfor 14 days. On day 15, one hour after treatment, the hypoxiatime [14] was recorded individually for each animal by placingthe animal in an empty glass jar of 300-mL capacity attached toan electronic watch, and the time until onset of convulsion wasrecorded as the end point.Passive avoidance test: Male Wistar rats were used for the study.KG (100mg/kg, p.o.) was used as a standard. Test drug was ad-ministered for a period of 14 days at the graded doses of 25, 50,100, and 200mg/kg, p.o. Scopolamine (3mg/kg, i.p) was admin-istered in all the groups 5min prior to the first trial to inducememory deficit (amnesia). The animals were exposed to the firsttrial 1 h after the last administration of LPPM/A003 on day 15[15].Chronic restraint stress:Male Swiss albino mice, 10–12 weeks oldand weighing about 20–22 grams, were employed for this study.Mice were restrained in 50mL conical polypropylene tubes for12 h during the dark cycle (2000–0800 h) for 14 days [16]. Exper-imental animals were divided into seven groups of eight animalseach. Test drug was administered daily for the duration of the ex-periment, i.e., for 14 days at the dose levels of 25, 50, 100, and200mg/kg p.o. Group 8 received LPPM/A003 per se (200mg/kg)without any stress conditions.Lymphocyte immunophenotyping: On day 14, blood was takenfrom the retro-orbital plexus of animals from all the groups forthe assessment of various immune cells surface markers. FITC-la-beled CD4+ (T helper cells), PE-labeled CD8+ (T cytotoxic cells),and CD244+ (natural killer cells) murine monoclonal antibodieswere added directly to 100 µL of whole blood, which was thenlysed using whole blood lysing reagent (BD Biosciences). Follow-ing the final centrifugation, samples were resuspended in phos-phate buffer saline (pH, 7.4) [17] and analyzed directly on theflowcytometer (BD Biosciences) using Cell Quest Pro Software(BD Biosciences).Intracellular cytokine estimation: 100 µL of the whole blood wastaken in the different falcon tubes. Magnetic-bead assisted cellsorting (MACS) separated the CD4+ T and CD8+ T cells. With thistechnique, specific cells that have antibody-coated micro beadsare separated under a strong magnetic field from the whole cellsuspension according to their cell surface antigen. They werethen analyzed for intracellular IL-2 (CD4+ T cells), IL-4 (CD4+ Tcells), and IFN-γ (CD4+ T cells) as described [18].Estimation of corticosterone in LPPM/A003 treated mice: Cortico-sterone was assessed in animals subjected to restraint stress toobserve the effect of test drugs on plasma corticosterone levelsas corticosterone is the final effector of HPA axis activation dur-ing stress exposure [19]. On day 14, blood was collected and theplasma was separated. The corticosterone assay was performedby a competitive immunoenzymatic method (Elisa kit; NeogenCorporation). All samples were assayed in triplicate at a wave-length of 450 nm.Estimation of corticosterone in gallic acid treated mice: Gallic acid(GA), a major constituent of the extract, at the dose levels of 0.5,1, 2, and 4mg/kg was also tested for its corticosterone loweringactivity in restraint stressed animals. The same protocol of re-straint stress and corticosterone estimation was followed.Body and organ weights: After the last stress session, the bodyweights of all the animals from all the groups were taken. The an-

imals were subsequently sacrificed and their thymus, adrenalglands, and spleen, were removed and weighed.Evaluation of biochemical parameters: After final stress exposure,blood was collected from all the animals, and serum was sepa-rated for the estimation of alanine aminotransferase (ALT) [22],and alkaline phosphatase (ALP) [21]. After this, these animalswere sacrificed; their livers were quickly excised, cleaned of ad-hering tissue, weighed, and homogenized in chilled phosphatebuffer saline for the analysis of lipid peroxidation [22] and GSH[23] (Table 2S, Supporting information).

Statistical analysisThe results were subjected to statistical analysis using ANOVAwith post-Bonferroni test and expressed as the mean ± S.E.M.;** p < 0.01; * p < 0.05; ns – not significant. The flowcytometry da-ta analysis and the data presentation were performed using CellQuest Pro software.

Supporting informationData on the effect of LPPM/A003 on restraint stress-induced alter-ations in biochemical parameters, on body weights (g) in normalcontrol (unstressed), RST control (stressed), and LPPM/A003treated mice, on changes in spleen weight (atrophy) underchronic stress exposure for control, stress, and drug treatedgroups, HPLC graph of standard gallic acid and pyrogallol, calibra-tion curve for gallic acid and pyrogallol, and HPLC graph of LPPM/A003 with separation of gallic acid and pyrogallol are available asSupporting Information.

Results!

Pretreatment of mice with LPPM/A003 for 15 days increased theswimming endurance of mice (l" Table 1) and the most signifi-cant effect was observed at the oral doses of 100 and 200mg/kgwith percent increases in swimming time of 63.74% and 62.51%as compared to the control group. Similar results were obtainedfor physical stress-induced neuromuscular incoordination sinceLPPM/A003 exhibited significant antifatigue activity in a dose-dependent manner (l" Table 2). It also increased the hypoxia timein mice (l" Table 3) and reversed the scopolamine induced mem-ory deficit in rats (l" Fig. 2) thereby showing the potent adapto-genic potential of the test material.In case of the immunological alterations induced by chronicstress, the percentage of CD4+ and CD8+ T lymphocytes, and NKcells (CD244+) decreased significantly in the restraint controlgroup on the 14th day of restraint stress. CD4+ T cell count de-creased from 22.84 ± 0.68 (mean ± S.E.M., normal control) to14.92 ± 0.31 (restraint stress control group) and CD8+ cells de-creased from 14.61 ± 0.33 (normal control) to 9.18 ± 0.51 (stresscontrol). LPPM/A003 exhibited a dose-dependent increase withmaximum response being elicited at a 100mg/kg dose withCD4+ T cells count of 31.13 ± 0.44, CD8+ T cellscount of 21.56 ±0.43 (l" Fig. 3a and 3b), and CD 244+ cells count of 4.51 ± 0.16(l" Fig. 4a and 4b).The above results were further supported by intracellular cyto-kine estimation in which flowcytometric analysis revealed a sig-nificant decrease in the expression of CD4 specific intracellularIFN-γ, IL-2 (Th-1 cytokines), and IL-4 (Th2 cytokine) in stress con-trol animals. Percentage of IL-2 decreased from 9.23 ± 0.53 in thenaive control to 3.85 ± 0.45 in the RST-control group and IFN-γdecreased from 6.79 ± 0.24 (naive control) to 2.40 ± 0.45 (RST-


bTable 1 Effect of different dosesof LPPM/A003 on swimming en-durance time in mice. Results arerepresented as means ± S.E. withn = 6 in each group.

Treatment Dose

(mg/kg, p.o)

Swimming endurance

(in minutes; mean ± S.E)

% Activity against

normal control

Control + stress – 147.25 ± 3.20 –

KG + stress 100 201.25 ± 5.13 36.67↑*

LPPM/A003 + Stress 25 192.23 ± 5.20 30.54↑*

LPPM/A003 + Stress 50 214.72 ± 4.30 45.82↑*

LPPM/A003 + Stress 100 241.12 ± 4.30 63.74↑**

LPPM/A003 + Stress 200 239.30 ± 5.30 62.51↑**

↑: Percent increase in swimming endurance; asterisks represent a significant difference between the RST group and LPPM/A003 treated

groups; **p ≤ 0.01, *p ≤ 0.5. The statistical test employed was one-way ANOVA. KG – Korean ginseng, LPPM/A003 – Labisia pumila

Table 2 Effect of LPPM/A003 onneuromuscular coordination andfatigue in mice subjected to swim-ming induced stress. Antifatigueand muscle coordination effectwas expressed as the number ofmice which stayed on the rota-rodmore than 180 seconds (consid-ered untired with full motor coor-dination). The % effect of eachgroup was determined by quantalresponse, i.e., all-or-nonresponse ina group (n = 8).

Groups Dose (mg/kg, p.o.) Antifatigue effect (percent)

Immediate After 30 mins

1.Normal control (without stress) – 100 100

2. Stress control – 0 25a**

3. KG + stress 100 62.50b** 75 b**

4. LPPM/A003 + stress 25 12.50 bns 25 bns

5. LPPM/A003 + stress 50 37.50 b* 37.50 b*

6. LPPM/A003 + stress 100 62.50 b* 87.50 b**

7. LPPM/A003 + stress 200 37.50 b* 87.50 b**

Asterisks with p value “a” indicate comparison with NC and “b” indicates comparison with RST; ** p ≤ 0.01, * p ≤ 0.5. The statistical test

employed was ANOVA followed by post-Bonferroni test.

Table 3 Effect of different dosesof LPPM/A003 on acute hypoxiatime in normal mice.

Treatment Dose (mg/kg, p.o.) Hypoxia time

(in seconds; mean ± S.E)

% Activity against

normal control

Normal control – 989.50 ± 0.45 –

KG 100 1798.13 ± 0.48 81.72↑**

LPPM/A003 25 1568.60 ± 0.64 58.53↑*

LPPM/A003 50 1651.12 ± 0.56 66.86↑**

LPPM/A003 100 1768.56 ± 0.48 78.73↑**

LPPM/A003 200 1772.86 ± 0.38 79.16↑**

↑: Percent increase in swimming endurance. Results are expressed as the means ± S.E. with n = 6 in each group. Asterisks represent a

significant difference between the RST group and LPPM/A003 treated groups; ** p ≤ 0.01, * p ≤ 0.5. The statistical test employed was one-

way ANOVA analysis. KG – Korean ginseng, LPPM/A003 – Labisia pumila

Fig. 2 Bar diagram representing the changes in transfer latency time forcontrol, scopolamine, and drug treated groups. The drug treated groupwas compared with the scopolamine treated control group. Results arerepresented as means ± S. E. with n = 5 in each group. Asterisks with p value“a” indicate comparison with NC and “b” indicates comparison with sco-polamine control p value; ** p < 0.01; * p < 0.05. The statistical test em-ployed was ANOVA followed by post-Bonferroni test.

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control) (l" Fig. 5a to c). A decrease in the expression of IL-4 wasalso observed from 6.31 ± 0.39 in the naive control to 2.27 ± 0.31in the RST group (l" Fig. 6a and 6b). A dose-dependent increasewas observed in cytokines levels in the mice treated with gradeddoses of LPPM/A003 with themost significant increases observedat a dose level of 100mg/kg: an increment in IFN-γ expression of7.03 ± 0.27 and for IL-2of 12.32 ± 0.83. Expression of IL-4 also in-creased in the LPPM/A003 treated group, although this increasewas not statistically significant.LPPM/A003 exhibited a significant antistress activity by lesseningthe chronic stress-induced raised corticosterone levels in a dose-dependent manner (l" Fig. 7a) and also considerably attenuatedstress-induced atrophy of spleen and thymus glands and hyper-trophy of adrenal glands (Fig. 1S, Supporting Information). To de-lineate the potential active constituent responsible for the ob-served activities, extensive screening of the identified com-pounds was carried out. Gallic acidwas found to possess themostsignificant corticosterone lowering activity (l" Fig. 7b) therebysuggesting its major role in the observed activities of the extract.Exposure to chronic restraint stress caused a significant altera-tion in hepatic function, increased the hepatic lipid per oxidation,

Fig. 3a Bar graphs represent the effect of graded doses of LPPM/A003 onstress-induced depletion of CD4+ and CD8+ T lymphocytes population.Results are represented as means ± S.E. with n = 8 in each group. NC – nor-mal control, RST – restraint stress control, KG – Korean ginseng, LPPM/A003 – Labisia pumila. LPPM/A003 had no per se effect on the experimentalindices. Asterisks with p value “a” indicate comparison with NC and “b” in-dicates comparison with RST; ** p ≤ 0.01, * p ≤ 0.5. The statistical test em-ployed was ANOVA followed by post-Bonferroni test.

Fig. 3b Flowcytometry scatter plots for T cell populations (CD4+ T helpercells subset and CD8+ cytolytic/cytotoxic T cells). The plots representevents for one representative mouse from each group of normal control,restraint stress control, KG and LPPM/A003 treated group. The gating andthe quadrants are set according to the standard procedures of the BD‑LSRflowcytometer.

Fig. 4a Bar graphs representing the effect of LPPM/A003 on stress-in-duced depletion of CD244+ NK cell population. Results are represented asmeans ± S.E. with n = 8 in each group. NC – normal control, RST – restraintstress control, KG – Korean ginseng, LPPM/A003 – Labisia pumila. Asteriskswith p value “a” indicate comparison with NC and “b” indicates comparisonwith RST; ** p ≤ 0.01, * p ≤ 0.5. The statistical test employed was ANOVAfollowed by post-Bonferroni test. LPPM/A003 had no per se effect on theexperimental indices.

Fig. 4b Flow cytometric scatter plots for CD244+ T lymphocytes popula-tion. The plots represent events for one representative mouse from eachgroup of normal control, RST-control, KG and LPPM/A003 treatedmice. Thegating and the quadrants are set according to the standard procedures ofthe BD‑LSR flowcytometer.

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band decreased the GSH (glutathione) significantly. It also in-
creased the serum levels of ALT and ALP. However, treatment ofanimals with LPPM/A003 reversed these altered values in adose-dependent manner. The significant results were obtainedat the dose levels of 100mg/kg and 200mg/kg. KG (100mg/kg)used as a standard also altered these hepatic and serum parame-ters significantly (Table 2S, Supporting Information).

Discussion!

In response to stressful stimuli, the HPA axis is activated resultingin hypersecretion of glucocorticoids, cortisol in humans and cor-ticosterone in rodents, which is mainly responsible for severalstress related changes in the body like inhibition of cellular andhumoral immune responses [24]. Flow cytometric analysis re-vealed that chronic restraint stress caused significant reduction

in the T lymphocytes population along with a marked decreasein the expression of IL-2 (a growth factor for T cells), IFN-gamma(a signature cytokine for Th1 response), and IL-4 (a Th2 specificcytokine). The treatment of animals with LPPM/A003 reversedthe effect of restraint stress on these cell types and related cyto-kines showing it to have efficient antistress potential. Duringstress, nerve terminals accelerate the recruitment of lympho-cytes to blood from the spleen which is the major storage poolof lymphocytes [25]. This results in the squeezing of the spleencausing a reduction in weight as observed in chronic stress expo-sure. Also persistent high levels of corticosterone during chronicstress causes apoptosis and necrosis in immature T and B cells re-sulting in the decline of thymus weight [26]. But the treatment ofanimals with LPPM/A003 significantly reversed the chronicstress-induced adrenal hypertrophy and atrophy of the spleenand thymus in a dose-dependent manner with the most signifi-cant effect at 100mg/kg. It also normalized the chronic stress-in-


bFig. 5a Effect of LPPM/A003 on CD4 specific in-tracellular IL-2 and IFN gamma expression in chroni-cally stressed mice. NC – normal control, RST – re-straint stress control, KG – Korean ginseng, LPPM/A003 – Labisia pumila. Results are represented asmeans ± S.E. with n = 8 in each group. Asteriskswith p value “a” indicate comparision with NC and“b” indicates comparison with RST; ** p ≤ 0.01,* p ≤ 0.5. The statistical test employed is ANOVAfollowed by post-Bonferroni test.

Fig. 5b and c Flow cytometric histogram repre-sentation for CD4 specific intracellular IL-2 and IFNgamma expression, respectively. The histogramrepresents events for one representative mousefrom each group of normal control, RST-control, KGand LPPM/A003 treated mice. The gating and thequadrants are set according to the standard proce-dures of the BD‑LSR flowcytometer.

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Fig. 6a Effect of LPPM/A003 on CD4 specific in-tracellular IL-4 expression in chronically stressedmice. NC – normal control, RST – restraint stresscontrol, KG – Korean ginseng, LPPM/A003 – Labisiapumila. Results are represented asmeans ± S.E. withn = 8 in each group. Asterisks with p value “a” indi-cate comparison with NC and “b” indicates com-parison with RST; **p ≤ 0.01, *p ≤ 0.5. The statisti-cal test employed was ANOVA followed by post-Bonferroni test.

Fig. 6b Flow cytometric histogram representationfor CD4 specific IL-4 expression. The histogram rep-resents events for one representative mouse fromeach group of normal control, RST-control, KG andLPPM/A003 treated mice. The gating and the quad-rants are set according to the standard proceduresof the BD‑LSR flowcytometer.

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bduced increase in serum alanine aminotransferase (ALT), alkaline
phosphatase (ALP), and hepatic lipid peroxidation (LP) levels aswell as augmented the CS induced decrease in hepatic gluta-thione (GSH). Thus, our results clearly indicate that Labisia pum-ila, a reputed plant in the Malaysian traditional system of medi-cine, possesses a considerable stress lowering potential. Afterscreening of the identified compounds, we found that gallic acidmight account for the observed biological properties as it has alsoshown significant corticosterone lowering activity along with theparent extract in stressed animals.Swimming endurance reflects physical endurance [27] and great-er swimming endurance has been reported in mice when pre-treated with antistress agents [28]. Our results also suggest thatthe test material has the potential to increase physical enduranceas well as overall performance in mice. It also enhanced the hyp-oxia time and showed recovery of physical stress-induced deple-tion of neuromuscular coordination. Stressful conditions ad-versely affect the cholinergic system and result in learning defi-cits [29]. There is substantial clinical evidence that muscarinic re-

ceptor blockade by drugs like scopolamine results in disruptionsof working (short term) memory, retrieval from reference (longterm memory) and strategy selection, and altered sensory proc-essing [30]. In our study mice treated with LPPM/A003 exhibiteda significant increase in TL (retention) even after treatment withscopolamine thus demonstrating the potential antiamnesic effectof LPPM/A003.To conclude, our results indicate that LPPM/A003 possesses apromising antistress/adaptogenic potential, as shown by its miti-gating effect on stress-induced behavioral and biochemical alter-ations, and also reverses the impact of chronic stress on T cellfunction and corresponding cytokines thereby counteracting thechronic stress-induced suppression of the immune system.

Acknowledgements!

The authors are thankful to Dr. Rajen and Ms. Lai Phung, HolistaBiotech, Malaysia, for the supply of plant material.


Fig. 7a Effect of LPPM/A003 on plasma cortico-sterone levels in chronically stressed mice. NC –normal control, RST – restraint stress control, KG –Korean ginseng, LPPM/A003 – Labisia pumila. Re-sults are represented as means ± S.E. with n = 8 ineach group. Asterisks with p value “a” indicatecomparison with NC and “b” indicate comparisonwith RST; ** p ≤ 0.01, * p ≤ 0.5. The statistical testemployed was ANOVA followed by post-Bonferronitest.

Fig. 7b Effect of GA on plasma corticosteronelevels in chronically stressed mice. NC – normalcontact, RST – restraint stress contact, GA – gallicacid. Asterisks with p-value “a” indicate comparisonwith NC and “b” indicate comparison with RST;** p ≤ 0.01, * p ≤ 0.5. The statistical test employedwas ANOVA followed by post-Bonferroni test.

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