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IMMUNOMODULATORYACTIVITYOFSAPONINMIXTUREISOLATEDFROMLAGENARIASICERARIAFRUITS

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43Nig. J. Nat. Prod. and Med. Vol. 12 2008

IMMUNOMODULATORY ACTIVITY OF SAPONIN MIXTURE ISOLATED FROMLAGENARIA SICERARIA FRUITS.

Gangwal A.1*, Parmar S.K.1, Mardia R.B.2, Sheth N.R.1

1 Department of Pharmaceutical Sciences, Saurashtra University, Rajkot 360005, India, 2 N. R.Vekaria Institute of Pharmacy andResearch Centre, Junagadh 362001, India ,* Corresponding author, Email:gangwal.amit@gmail.com, Phone no.:

09375927522, Address: 1635- B, Scheme no. 71, Behind ranjit hanuman temple, Indore- 452009(M.P.), India

Key words: Immunomodulation, Lagenaria siceraria fruits, Delayed type hypersensitivity, Humoral antibody titre

ABSTRACTAn attempt has been made to assess the immunomodulatory activity of purified saponin mixture (PSM) isolated from the fruits of Lagenaria siceraria atthree dose levels ranging from 50-150 mg/kg body weight in mice using haemagglutination antibody (HA) titre, delayed type hypersensitivity (DTH) andcarbon clearance parameters. PSM increased haemagglutination antibody titre and carbon clearance significantly but inhibited delayed type hypersensitivityresponse in a dose dependent manner. Thus, present study, for the first time, demonstrated the immunomodulatory activity of Lagenaria siceraria fruits.

INTRODUCTION

Lagenaria siceraria (Molina) Standley (family Cucurbitaceae),commonly known as lauki (Hindi) and bottle gourd (English), is amedicinal plant and utilizable species [1]. The plant enjoys thereputation of being one of the earliest plants to be domesticatedon earth [2]. It is a climbing or trailing herb with bottle, oval ordumb bell shaped fruits [3]. Lagenaria siceraria fruits aretraditionally used for its cardioprotective, cardiotonic, general tonicand aphrodisiac properties [4]. They are also used in the treatmentof various allergic and inflammatory disorders like bronchialasthma, rhinitis, bronchitis [5] and rheumatism [6]. Various extractsof fruits of Lagenaria siceraria were found to have anti-inflammatory, analgesic [7], hepatoprotective [8],antihyperlipidemic [3], diuretic [9] and antibacterial activities [10].These properties of Lagenaria siceraria fruits have beenattributed to its isolates namely saponins, carbohydrates andflavonoids [11].

Several studies have shown that therapeutic activity of variousextracts of higher plants may be mediated by interaction with thehost’s immune system and saponins, may in part, contribute totheir medical uses [12, 13]. Saponins have long been recognizedto possess a wide variety of biological activities. Several saponinglycosides, isolated from medicinal plants, have been discoveredto possess significant immunomodulatory effects [14]. Saponinshave shown stimulatory effects on the components of specificimmunity and on monocyte proliferation. Quillaia and othersaponins either as crude mixture or as purified compounds havebeen reported to increase immune–cell proliferation in vitro andboost antibody production. Ginseng and Astragalus are otherplants having saponins as their major constituents with provenimmunomodulatory activity [15].

Saponins from other plants of Cucurbitaceae family have beenreported to possess immunomodulatory activity [16]. It will be ofinterest to know the immunomodulating activities of the purifiedsaponin mixture (PSM) of Lagenaria siceraria fruits. Moreover,combination of ethnopharmacological reports and recent

experimental data seem to indicate an involvement of the immunesystem in the therapeutic action of Lagenaria siceraria fruits,clarification of its mechanism of action needs further studies. Thepresent paper reports pioneer investigation on the effect of PSMisolated from Lagenaria siceraria fruits on cellular and humoralimmune responses and on in vivo phagocytosis.

RESULTSPresence of saponins in PSM was confirmed by the interpretationof the color reactions with vanillin–sulphuric acid and LiebermannBurchard’s reagent. Four violet colored spots having Rf values0.2, 0.34, 0.38 and 0.6 were seen on the plate sprayed with vanillinsulphuric acid while plate sprayed with Liebermann Burchard’sreagent showed three spots of reddish brown color having Rfvalues 0.22, 0.31 and 0.35. PSM answered the foam and hemolytictest confirming the presence of saponins.

The haemagglutination antibody (HA) titre was used to assesshumoral immune response. A dose dependent increase in bothprimary and secondary antibody titres was observed in mice treatedwith PSM as compared to control group animals (Table 1).

Cell mediated immune response was assessed by Delayed TypeHypersensitivity (DTH) reaction i.e. foot pad reaction comparedto control group animals. PSM produced a significant dosedependent decrease in DTH response in mice. Decrease in DTHreaction in response to Ship Red Blood Cells (SRBCs) revealedthe inhibitory effect of saponins on T cells (Table 2).

Rate of carbon clearance is the measure of competency of thereticuloendothelial system and its granulopoetic activity, the fasterremoval of carbon particles has been correlated with the enhancedphagocytic activity. In the present study an increased phagocyticactivity was observed in treated groups as compared to controlgroup animals in a dose dependent manner.

Nig. J. Nat. Prod. and Med. Vol. 12 200844

Table 1: Effect of PSM isolated from Lagenaria siceraria fruits on SRBC-induced haemagglutination antibodytitre in mice

Group Treatment Mean haemagglutinationantibody (HA) titre

Range 10 HA titre Range 20 HA titre

Control(sensitized) - 32-64 37.33±5.33 0-32 32.0±0.00

PSM treated 50 mg/kg 32-64 53.33±6.74 a 32-64 37.33±5.33 a

PSMtreated 100 mg/kg 64-128 117.33±10.66 b 64-128 85.33±13.49 c

PSM treated 150 mg/kg 64-256 202.67±34.72 c 32-128 90.67±17.36 c

Values are mean ± S.E.M, aP>0.05, bP<0.05, cP<0.01 vs control group animals, (n=6), 10 primary, 20

secondary

Table 2: Effect of PSM isolated from Lagenaria siceraria fruits on SRBC -induced delayed typehypersensitivity in mice

Group Treatment Foot pad thickness Inhibition % (mean % edema) (compared to control)

After 24 h After 48 h After 24 h After 48 h

Control(sensitized) - 63.41±1.43 43.12±0.32 - -

PSM treated 50 mg/kg 31.96±1.21c 24.43±1.11 c 49.59 43.34

PSM treated 100 mg/kg 18.27±0.52 c 14.12±0.37 c 71.18 67.25

PSM treated 150 mg/kg 14.45±0.76 c 10.80±0.43 c 77.21 74.95

Values are mean ± S.E.M, cP<0.01 vs control group animals, (n=6)

DISCUSSIONModulation of immune response through stimulation orsuppression may help in maintaining a disease-free state [23].Apart from being specifically stimulatory or suppressive, certainagents have been shown to possess activity to normalize ormodulate pathophysiological processes and hence calledimmunomodulatory agents [18].

In the present investigation, purified saponin mixture was isolatedfrom Lagenaria siceraria fruits. Different chemical tests wereemployed to confirm the presence of saponins in PSM. The mixturewas further subjected to thin layer chromatography to confirmthe presence of saponins, by the interpretation of color reactionafter spraying the plate with specific reagents. PSM was studiedfor its effect on HA titre, DTH response and in vivo phagocytosisin mice. The results obtained in the present study showed thatthe mixture displays a dose dependent immunomodulatory effects.

Injecting mice with 108 SRBCs suspended in saline sensitizes themfor elicitation of DTH response and also induces antibodysynthesis, therefore this system has major advantages i.e. itenables two arms of immune system to be measured in the samespecies under ideal condition and is relatively simpler to perform[21].

Antibody molecules, a product of B-lymphocytes and plasma cells,are central to humoral immune response; IgG and IgM are the

major immunoglobulins which are involved in the complementactivation, opsonization and neutralization of toxins etc. [23]. Theantibody production of T-dependent antigen, SRBCs, requiresthe co-operation of T-and B–lymphocytes and macrophages. Theaugmentation of humoral response by PSM, as evidenced by anenhancement of antibody responsiveness to SRBC in mice as aconsequence of both pre and post immunization PSM treatment,indicates the enhanced responsiveness of macrophages and B-lymphocytes subsets involved in antibody synthesis [24].

DTH response is antigen specific and causes erythema andinduction at the site of antigen injection in immunized animals.The histology of DTH response can be different for differentspecies, but the general characteristics are an influx of immunecells at the site of injection, and induction becomes apparent within24-72 hours. T-cells are required to initiate the reaction [18].

The interaction of sensitized T-cell with presented antigen isknown to be associated with the release of mediators such ashistamine, products of arachidonic acid metabolism and eventuallyinterferon–ã leading to delayed type hypersensitivity [25].Therefore, the inhibitory effect of PSM on DTH response couldbe due to their influence on the biological mediators. It is inaccordance with the previously published data on anti-inflammatory activity of Lagenaria siceraria fruits [7]. DecreasedDTH reactivity may further be explained by simultaneous presence

45Nig. J. Nat. Prod. and Med. Vol. 12 2008

of high titres of antibodies, promoting the elimination of antigenby non–presenting phagocytes [25].

Phagocytosis by macrophages is important against microorganismand its effectiveness is markedly enhanced by opsonization ofparasite with antibody and the complement C3b (opsonin) leadingto more rapid clearance of parasite from the blood [18].Administration of saponin mixture enhanced the carbon clearancerate from circulation in mice significantly, compared to the animalsof control groups.

To the best of our knowledge, for the first time, we reported theimmunomodulatory property of the fruits of Lagenaria siceraria.The present study with Lagenaria siceraria saponins leads us toconclude that these saponins suppress cellular immune response,augment humoral immune response and activate macrophageinduced phagocytosis. A bioassay guided fractionation of thesaponin mixture is now in progress to characterize and identifythe bioactive substance(s).

MATERIALS AND METHODS

Isolation of saponins from plant materialThe fresh fruits of Lagenaria siceraria were collected fromoutfield of Junagadh city, Gujarat state, India in March 2007. Plantwas authenticated by the authority of department of botany,Bahauddin College, Junagadh, where a voucher specimen (BS/Bot./I-5/06-07) has been deposited for future reference. Fruits werecut into pieces and dried. The coarsely powdered plant material(500 g) was then defatted with petroleum ether and then extractedwith methanol. The methanol extract was concentrated by distillingoff the solvent and evaporated to dryness using water bath. Theresidue (69 g) was suspended in water, extracted successivelywith ethyl acetate and n-butanol (3×300 ml each) and the resultingfractions were concentrated to provide ethyl acetate-soluble (3.2g), n-butanol-soluble (14.5 g) and water-soluble (47.3 g) portions.The n-butanol-soluble portion was subjected to glass columnchromatography and eluted with methanol to give PSM (7 g).

Chemical characterizationThin layer chromatography (silica gel 60 F254, layer thickness 0.2mm) was used to confirm the presence of saponins. The solventsystem used was chloroform:methanol (90:10 v/v). After sprayingthe plates with vanillin–sulphuric acid and Liebermann Burchard’sreagent separately, they were heated at 100º C for 10 minutes tovisualize the spots. PSM was also subjected to qualitative chemicaltests meant for identification of saponins [17].

Acute toxicity studyOver night fasted mice weighing (20-25 g) were used for the study.PSM was dissolved in normal saline and administered orally todifferent groups (each of five animals) in increasing dose levels of50, 100, 150, 250, 500, 1000 and 2000 mg/kg body weight. Animalswere observed closely for 1 hour, then frequently for 24 hoursthereafter once daily for 14 days [8]. During this period, animalswere observed for gross behavioral and morphological changes.No toxic symptoms were seen and all the animals survived thetoxicity studies at all dose levels. Based on the study, doses of 50,100 and 150 mg/kg were selected for animal experiments.

Experimental animalsSwiss albino mice (20-25 g) of either sex were used. The experimentalprotocols were approved by Institutional Animal EthicsCommittee. Mice were housed under standard environmentalconditions and had free access to standard pellet diet (Pranavagro sales, Ahmedabad, India) and water ad libitum. PSM wasdissolved in normal saline and administered orally for theevaluation of immunomodulatory activity in mice. Control groupanimals received only normal saline.

AntigenFresh blood was collected from sheep sacrificed in local slaughterhouse into Alsever’s solution. During the experiment, adequateamount of stock solution of Sheep red blood cells (SRBCs), storedin Alsever’s solution, was taken and allowed to stand at roomtemperature. It was washed three times with normal saline. Thesettled SRBCs was then suspended in normal saline and RBCs ofthis suspension were adjusted to a concentration of 5x109/ml forimmunization and challenge [18].

SRBC–induced humoral antibody (HA) titreThe method described by Atal et al., 1986 [19] was followed. Groupsof six mice per treatment were immunized by injecting 20 μl ofSRBC suspension (5x109 SRBC/ml) subcutaneously into right hindfoot pad. Seven days latter they were challenged by injecting 20μl of SRBC suspension (5x109 SRBC/ml) intradermally into the lefthind foot pad. The day of immunization was referred to as day 0.Blood samples were collected from all the animals separately byretroorbital puncture on day +7 (before challenge) for primaryantibody titre and on day +14 for secondary antibody tire.Antibody levels were determined by the method described byShinde et al., 1999 [20]. Briefly 25 μl aliquot of serum of eachanimal was placed on microtitre plates. To serial two–fold dilutionsof pooled serum (made in 25 μl normal saline), 25 μl of 1% v/vSRBC suspension (in normal saline) was added. The microtitreplates were kept at room temperature for 1 h and then observedfor haemagglutination (until control wells showed unequivocallynegative pattern). The value of the highest serum dilution showinghaemagglutination was taken as the antibody titre. The PSM wasadministered orally once daily, starting with 7 days prior tosensitization till the challenge (-7, -6, -5, -4, -3, -2, -1, 0, +1, +2, +3,+4, +5, +6, +7).

SRBC–induced delayed type hypersensitivity (DTH)Delayed type hypersensitivity response was induced in mice bythe method of Doherty, N.S., 1981 [21]. Groups of six mice pertreatment were immunized by injecting 20 μl of SRBC suspension(5x109 SRBC/ml) subcutaneously into the right hind foot pad ofeach mouse. The day of sensitization was designated as day 0.Seven days latter, the thickness of the left hind foot pad wasmeasured using a vernier caliper reading to 0.01 mm. The micewere then challenged by injecting 20 μl of SRBC suspension (5x109

SRBC/ml) intradermally into the left hind foot pad. Foot padthickness was measured again 24 and 48 hours after challenge.The difference between the pre and post challenge foot padthicknesses (expressed in mm) were taken as a measure of DTHresponse and results were expressed as percent change in

Nig. J. Nat. Prod. and Med. Vol. 12 200846

thickness. The PSM was administered orally once daily, startingwith 7 days prior to sensitization till the challenge (-7, -6, -5, -4, -3,-2, -1, 0, +1, +2, +3, +4, +5, +6, +7).

In vivo phagocytosisIt was performed by the method described by Jayathirtha et al.,2004 [22]. Animals of the different treatment groups were givenPSM (50-150 mg/kg) daily for 5 days. At the end of five days, after48 hours, mice were injected via the tail vein with carbon inksuspension (10 μl/gm body weight) (Pelican Germany). Bloodsamples were drawn (in EDTA solution, 5 μl) from the retroorbitalvein at 0 and 15 minutes. A 25 μl sample was mixed with 0.1%sodium carbonate solution (2 ml) and its optical density wasmeasured at 680 nm. The phagocytic index (K) was calculatedusing the equation: K= (log OD1-log OD2)/15 Where OD1 and OD2are optical densities (OD) at 0 and 15 minutes respectively.

Statistical methodsDifference between experimental and control groups were analyzedby the student’s t-test. Data was presented as mean values ±S.E.M. and P<0.05 was considered as statistically significant.

ACKNOWLEDGEMENTWe owe our thanks to Dr. M.M. Jani, Head, Department of Botany,Bahauddin Science College, Junagadh, for authentication of plantspecimen. The facilities provided by the Department ofPharmaceutical Sciences, Saurashtra University, Rajkot andfinancial assistance by GUJCOST, Government of Gujarat, duringthe course of this study are greatly acknowledged.

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