Effect of naturally occurring triterpenoids glycyrrhizic acid, ursolic acid, oleanolic acid and nomilin on the immune system. (2024)

Summary

The effect of naturally occurring triterpenoid compounds such asglycyrrhizic acid, ursolic acid, oleanolic acid, and nomilin on theimmune system was studied using Balb/c mice. Intraperitoneal treatmentswith 5 doses of these terpenoid compounds were found to enhance thetotal white blood cells (WBC) count. In ursolic acid, oleanolic acid andnomilin treated animals the maximum total WBC count was observed on the6th day, while in glycyrrhizic acid treated animals it was observed onlyon the 9th day after the drug treatment. In ursolic acid, oleanolic acidand nomilin treated animals the percentage of increase in the total WBCcount was to 91.48 [+ or -] 4.6%, 135.75 [+ or -] 6.4% and 117.33 [+ or-] 17.9% respectively. In the glycyrrhizic acid treated animals thetotal WBC count was increased to 114.9 [+ or -] 18%. Bone marrowcellularity and [alpha]-esterase positive cells were also enhanced bythe treatment with these terpenoids. Treatment with varioustriterpenoids along with antigen produced an enhancement in the specificantibody titre and the number of plaque forming cells (PFC) in thespleen. Triterpenoids remarkably inhibited delayed type hypersensitivityreaction (DTH). These results indicate the immunomodulatory activity ofnaturally occurring triterpenoids such as glycyrrhizic acid, ursolicacid, oleanolic acid and nomilin.

Key words: glycyrrhizic acid, ursolic acid, oleanolic acid, nomilinand immune system

* Introduction

Immune system is a very complex homeostatic system consisting of anetwork of interacting cells, tissues and organs. It allows the organismto exist within itself and maintains a surveillance mechanism torecognize certain components that are considered nonself.Immunomodulators are substances, which modify the activity of the immunesystem. Immunomodulators have biphasic effects some tend to stimulateimmune system which are low while inhibit host defense parameters whichare normal or already activated (Stites et al. 1980). Immunosuppression is a major draw back in conventional cancer therapies.

Plant and plant products have been the basis of treatment of humandiseases since time immemorial. Some of the plants with knownimmunomodulatory activity are Viscum album (Kuttan and Kuttan, 1992),Tinospora cordifolia (Mathew and Kuttan, 1999), Withania somnifera(Davis and Kuttan, 2000) and Echinacea speczies (Wagner, 1999).

Terpenes are the class of compounds widely distributed in plants.They occur widely in food consumed by humans (Steinmetz and Potter,1991). The limonoids, limonin and nomilin are oxidized triterpenes,which are responsible for bitter taste of citrus fruits. They have shownto inhibit azoxymethane induced colon carcinogenesis in rats (Tanaka etal. 2000). Although triterpenoids are widely used for medicinal purposesin many Asian countries, this class of molecules, which resemblessteroids in their chemical structures, biogenesis and pleiotropicactions has not impacted on the practice of western medicines (Suh etal. 1998). Triterpenoids like the steroids are formed in nature by thecyclisation of squalene with the retentions of all 30 carbon atoms inthe molecule such as oleanolic acid and ursolic acid. Oleanolic acid andursolic acid have known anti-inflammatory and anticarcinogenic activity(Deepak and Handa, 2000; Liu, 1995; Suh et al. 1998).

The effect of triterpenoids on immune system has not been studiedin a systematic way. The main aim of the study was to evaluate theeffect of naturally occurring triterpenoids such as glycyrrhizic acid,ursolic acid, oleanolic acid and nomilin (Fig. 1a, b, c, d) on theimmune system. Balb/c mice, which are highly immunogenic, were used tostudy the effect of these triterpenoids on the immune system. Theformulae of these triterpenoids compounds is given in Fig. 4.

[FIGURES 1 & 4 OMITTED]

In the present study we report the immunomodulatory activity oftriterpenoids such as glycyrrhizic acid, ursolic acid, oleanolic acidand nomilin by analysing their effects on hematological parametes,production of bonemarrow cells, production of specific antibody andantibody producing cells in spleen.

* Materials and Methods

Balb/c mice were taken from Amala Cancer Research Centre animalbreeding section. The animals were kept in air-controlled rooms fed withnormal mice chow (Sai Feeds, India) and water ad libitum.

Sheep red blood cells (SRBC) was collected from localslaughterhouse in Alsever's solution.

Triterpenoid compounds glycyrrhizic acid, ursolic acid, oleanolicacid and nomilin were purchased from Sigma Chemicals, USA.Pararosaniline and [alpha]-napthylacetate were obtained from LobaChemie, Bombay. Harris hematoxilline was purchased from Glaxo IndiaLtd., Bombay. All other chemicals were analytical reagent grade.

Drug preparation: Triterpenoids were suspended in light paraffinoil.

Experimental design: Six groups (7 nos/group) of Balb/c mice(male,4-6 weeks old, 20-25 g wt) were used in all the experiments. Group I II,III, and IV animals were treated intraperitoneally with glycyrrhizicacid, ursolic acid, oleonolic acid at concentration of 50[micro]moles/kg body wt/dose/animal and nomilin (10 [micro]moles/kg bodywt/dose/animal) respectively for 5 days. Group V was treated withvehicle (paraffin oil) and group VI was kept as untreated control.

All the animal experiments were performed according to the rules ofAnimal Ethics Committee, Govt. of India.

Determination of the effect of triterpenoids on the hematologicalparameters

The animals were treated with the terpenoid compounds as explainedabove. Blood was collected from the tail vein and various parameterssuch as total WBC count, (Haemocytometer) differential count(Leishman's stain) and Hb content (Cyanmet hemoglobin method) wererecorded prior to the drug treatment and continued every 3rd day for onemonth. The change in the body weight was also recorded.

Determination of the effect of triterpanoids on the bone marrowcellularity and [alpha]-esterase positive cells

The animals were sacrificed 24 h after the last dose of the drugtreatment, bone marrow cells from femur were collected and made into asingle cell suspension. The bone marrow cellularity was determined usinghaemocytometer.

The number of [alpha]-esterase positive cells were determined bythe azo dye coupling method (Bancroft and Cook, 1984). A smear of bonemarrow cells from the above preparation was made on clean glass slides,stained with [alpha]-naphthyl acetate and pararosaniline hydrochlorideand counter stained with heamatoxiline. The number of [alpha]-esterasepositive cells were expressed out of 4000 cells.

Determination of the effect of triterpenoids on production ofspecific antibody

All the animals were immunized with sheep red blood cells (20% SRBCin 0.1 ml) along with the first dose of drug administration. After the5th dose of drug treatment blood was collected from the tail vein andcontinued every third day for one month. Serum was separated and heatinactivated at 56 [degrees]C. Antibody titre was determined byhemagglutination assay using SRBC as antigen (Singh et al. 1984).

Determination of the effect of triterpenoids on PFC in spleen

After the 5 doses of drug treatment all the animals were immunized(i.p) with SRBC (2.5 x [10.sub.8] cells/20 [micro]l/animal). The animalswere sacrificed on different days, spleen processed, made to single cellsuspension and used for determining the antibody producing cells byJern's Plaque assay (Jerne and Nordin, 1963).

Determination of the effect of Triterpenoids on delayed typehypersensitivity (DTH) reaction

All the animals were immunized (i.p) with SRBC (1 x [10.sup.8]/20[micro]l/animal) in the presence and absence of terpenoid compounds. Thecompounds were administered on the same day of immunization andcontinued for 5 days. Along with the 5th dose of test compounds animalswere injected with a challenging dose of SRBC (1 x [10.sub.8]/20[micro]l/animal) on the left hind paw. DTH was determined by measuringthe thickness of paw 24 h after the challenging dose of the antigen(Langrange et al. 1974).

Statistical analysis

All the experiments were repeated twice and the results areexpressed as mean [+ or -] S.D. Statistical evaluation of the data wasdone using Student t-test.

* Results

Effect of triterpenoids on the hematological parameters

Administration of triterpenes inereased the total WBC count inBalb/c mice (Fig. 2). The maximum WBC count was obtained on 6th day inthe animals treated with oleanolic acid (135.75 [+ or -] 6.4%) followedby nomilin (117.33 [+ or -] 17.9%) and ursolic acid (91.48 [+ or -]4.6%). In the glycyrrhizic acid treated animals the maximum count wasobserved only on the 9th day (114.9 [+ or -] 18%).

[FIGURE 2 OMITTED]

Haemoglobin content was moderately increased by the administrationof triterpenoid compounds. There was no appreciable change in thedifferential count and body wt. (data not shown) after the terpenoidadministration.

Effect of triterpenoids on the bone marrow cellularity and[alpha]-esterase positive cells.

The effect of triterpenoids on the bone marrow cellularity and[alpha]-esterase positive cells is given in Table 2. Triterpenoidstreated group of animals showed a remarkable increase in bone marrowcell number compared to control animals. The maximum increase in thenumber of bone marrow cell was observed in animals treated witholeanolic acid (91.3 [+ or -] 8.6%) followed by nomilin (77.3 [+ or -]13%), ursolic acid (41.1 [+ or -] 4.7%) and glycyrrhizic acid (36.8 [+or -] 6.6%). The number of [alpha]-esterase +ve cells was significantlyincreased in animals treated with oleanolic acid (35.48 [+ or -] 6.3%)nomilin (23.37 [+ or -] 8.8%) ursolic acid (19.3 [+ or -] 2.3%) andglycyrrhizic acid (15.9 [+ or -] 5.4%) compared to the normal animals.

Effect of triterpenoids on production of specific antibody

The aim of this experiment is to check the specific immune responseby using sheep red blood cells (SRBC). There was a significant increasein the production of specific antibody in animals treated with theterpenoid compounds (Fig. 3). The maximum antibody titre of 1024 wasobserved in the nomilin, oleanolic acid and ursolic acid treated animalson 9th day after the antigen administration. The antibody titre remainedas 1024 till 15th day in nomilin treated animals. In glycyrrhizic acidtreated animals the maximum antibody titre 1024 was observed only on12th day.

[FIGURE 3 OMITTED]

Effect of triterpenoids on plaque forming cells (PFC) in spleen

The effect of terpenoid compounds on the number of plaque formingcells is shown in Fig. 4. The maximum number of plaque forming cells wasobserved in terpenoid treated animals on 5th day after immunization. Thepercentage of increase of PFCs on 5th day were 201 [+ or -] 3.1%(ursolic acid) 148.4 [+ or -] 5.4% (oleanolic acid) 111.13 [+ or -] 6.1%(nomilin) and 96.5 [+ or -] 2.2% (glycyrrhizic acid) compared withanimals which have received antigen alone.

Effect of triterpenoids on delayed type hypersensitivity (DTH)reaction

The effect of triterpenoids on delayed type hypersensitivity isgiven in Table 2. Triterpenes were found to inhibit the delayed typehypersensitivity reaction remarkably. The maximum inhibition (95%) ofDTH reaction was observed in glycyrrhizic acid treated group. Treatmentswith ursolic acid, oleanolic acid and nomilin could inhibit the DTHreaction by 76%, 88% and 76% respectively.

* Discussion

The immune system is known to be evolved in the etiology as well aspathophysiologic mechanism of many diseases. The use of immunomodulatorydrugs both to treat and prevent some diseases have been increasing. Thenumber of immunodefficient patients is growing constantly because ofstress, toxic factors, and administration of drugs that may adverselyinfluence the human immunological systems. Immunomodulation is theregulation of immune response whether to suppress them when unwanted orto stimulate them in the prevention of infectious diseases.Immunomodulatory agents that are free from side effects and which can beadministrated for long duration to obtain a continuous immune activationare highly desirable for the prevention of diseases. There are a varietyof naturally and chemically derived compounds discovered withimmunomodulatory activity such as levamasole, glucan, IL-2, IFN etc. areused in combination with cisplatin, adiramycin, 5-flurourasil etc.against many types of carcinomas. But most of these compounds have sideeffects namely fever, myalgias fatigue etc. (Herberman and Pinsky, 1985)The present study demonstrate that unlike other chemically definedcompounds, these triterpenoid compounds modulate the immune systemwithout affecting the other parameters of the body

The majority of all the cell type involved in the immune system isproduced from common hemopoietic stem cells of bone marrow. Bone marrowalso provides microenvironment for antigen dependent differentiation ofB cells. Administration of Withania somnifera, plant having someimmunopotentiating activity also increase the total bone marrow cells(Davis and Kuttan, 2000). The present study shows an enhancement in bonemarrow cellularity and total WBC count by the administration of variousterpenoids indicating their effect on the hematopoiesis. Both innate andadaptive immunity depends upon the activities of WBC. Terpenoids couldenhance the total WBC count on the 6th day. The innate and adaptiveimmune system together provides a remarkably effective defence system.

The mononuclear phagocyte system is composed of bone marrowmonocytes precursors, circulating monocytes and macrophages in alltissues including lung liver spleen, adrenal glands, intestine and bonemarrow. These cells are highly phagocytic and participate in theeradication of invading microorganisms and clearance of damaged orscenscent autologus tissues. They also play an active role in antigenprocessing and prevention to T lymphocytes to generates the primaryimmune response. Like proliferating bone marrow cells, bone marrowmonocyte show non-specific esterase activity (Hayhoe, 1980).Administration of these terpenoid compounds increased the number of[alpha]-esterase active bone marrow cells. This indicates the effect ofadministration of triterpenes on the stem cell proliferation anddifferentiation

The humoral immune response was analysed by the specific antibodyproduction and number of antibody producing cells in spleen. Theproduction of antibody producing cells in spleen was increased byterpenoid administration. The circulating antibody titre was alsosignificantly enhanced in the terpenoid treated animals; showing itsmodulatory effect on the humoral arm of the immune system.

T[H.sub.1] response stimulate the cytokine profile which activatemainly TC cells and macrophages where as T[H.sub.2] response activatemainly B cells. Immune response involved here is T[H.sub.1]response.Since delayed type hypersensitivity reactions are mediated byspecific T cells together with denitritic cells, macrophages, andcytokines especially interleukin 1. The overall effect of thesecytokines is to draw macrophages into the area and activate them,promoting increased phagocytic activity and increased concentration oflytic enzymes for more effective killings (Tami, 1986). As lytic enzymeleak out of the activated macrophages into surrounding tissues localizedtissue destruction can be ensured. These non-specific distructions ofcells in chronic DTH reaction, characterized by excessive number ofmacrophages continual release of lytic enzymes are not desirable.Certain plant originated compounds have the capacity to inhibit chronichypersensitivity reaction (Davis and Kuttan, 2000). Administration oftriterpenoids such as glycyrrhizic acid, ursolic acid, oleanolic acidand nomilin has been shown to inhibit the hypersensitivity reaction.Among of these compounds glycyrrhizic acid could inhibit the reactionnearly to 95%. Ursolic acid, oleanolic acid and nomilin could producebetween 75-85% inhibition of hypersensitivity reaction.

These results give clear evidence for the immuno modulatoryactivity of terpenoid compounds glycyrrhizic acid, ursolic acidoleanolic acid and nomilin. A detailed analysis on immunomodulatoryactivity of these naturally occurring terpenoids has to be carried out.

Table 1. Effect of triterpenes on bone marrow cellularity and[alpha]-esterase activity.Treatment Bone marrow ([alpha]-esterase activity cellularity (no: of [alpha]-esterase (cells/femur) +ve cell/4000 cells) x [10.sup.6]Normal Control 15.5 [+ or -] 2.3 1063 [+ or -] 69.4Vehicle 15.4 [+ or -] 0.43 1050 [+ or -] 58.5 (paraffin oil)Glycyrrhizic acid 22 [+ or -] 0.7 * 1232 [+ or -] 43.3 *Ursolic acid 20.1 [+ or -] 1.15 * 1360 [+ or -] 79.5 *Oleanolic acid 28.8 [+ or -] 1.08 * 1496 [+ or -] 245 *Nomolin 27.65 [+ or -] 1.95 * 1254.3 [+ or -] 97.38 *Balb/c mice were treated with five doses of triterpenoids.Bonemarrow cells were collected from femur and made intoa single cell suspension. The bone marrow cellularity wasdetermined using heamocytometre. The -esterase positivecells were determined by the azo dye coupling method. Plus-minusvalues are means [+ or -] SD. Student's t-test was used to compare themean cell counts compared with untreated normal animals (* P < 0.001).Table 2. Effect of triterpenoids on delayed typehypersensitivity reaction.Treatment Initial paw Paw thickness % of thickness (cm) after 24 hrs (cm) inhibition compared to Antigen aloneAntigen alone 0.18 [+ or -] 0.009 0.35 [+ or -] 0.02Paraffin oil (vehicle) 0.14 [+ or -] 0.01 0.33 [+ or -] 0.01 0%Glycyrrhizic 0.186 [+ or -] 0.01 0.194 [+ or -] 0.01 95% acidUrsolic acid 0.18 [+ or -] 0.01 0.22 [+ or -] 0.02 76%Oleanolic acid 0.20 [+ or -] 0.07 0.22 [+ or -] 0.01 88%Nomilin 0.19 [+ or -] 0.01 0.23 [+ or -] 0.01 76%All animals were senzitised with SRBC (1 x [10.sup.8] cells/20[micro]l) subcutaneously on ventral side. Treated animals receivedfive doses of various triterpenoids. One group kept as untreatedcontrol and another group as paraffin treated control animals.After the 5th dose, all the animals injected with challenging doseof SRBC (1 x [10.sup.8] cells/20 [micro]l) on left hand paw. DTHdetermined by measuring the paw thickness after 24 h.

Acknowledgement

The work was financed by a grant from the Committee for Science,Technology and Environment, Dept. of Planning and Economic Affairs,Govt. of Kerala. Authors would like to acknowledge Dr. R. Kuttan,Research Director, Amala Cancer Research Centre for his encouragement tocarryout this work.

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* Address

G. Kuttan, Ph.D., Amala Cancer Research Centre, Amalanagar,Thrissur, Kerala, India

Fax: 91 487 307868

e-mail: [emailprotected]

T.J. Raphael and G. Kuttan

Amala Cancer Research Centre, Amalanagar, Thrissur, Kerala, India