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Journal of Ethnopharmacology 131 (2010) 505508

Contents lists available at ScienceDirect

Journal of Ethnopharmacology

j o u r n a l h o m e p a g e : w w w . e l s e v i e r . c o m / l o c a t e / j e t h p h a r m

Ethnopharmacological communication

Modulating effect ofLeptadenia reticulata (Retz) Wight & arn against chromate

(VI)-induced immunosuppression and oxidative stress on mouse splenic

lymphocytes and bone marrow derived macrophages

V. Girishkumar a, M. Sreepriya b,, S. Praveenkumar b, Geetha Bali b, M.S. Jagadeesh a

a Department of PG Studies in Dravyaguna, Government Ayurveda Medical College, Bangalore-560 009, Karnataka, Indiab Department of Microbiology and Biotechnology, Bangalore University, Jnana Bharathi Campus, Bangalore-560 056, Karnataka, India

a r t i c l e i n f o

Article history:

Received 31 May 2009

Received in revised form 9 June 2010

Accepted 28 June 2010

Available online 7 July 2010

Keywords:

Leptadenia reticulata

Chromate (VI)

Experimental immunosuppression

Oxidative stress

Immunomodulation

a b s t r a c t

Ethnopharmacological relevance: Leptadenia reticulata (Retz) Wight & arn is mentioned in the ancient

ayurvedic literature as an immune booster and rejuvenator.

Aims of thestudy: To investigate,the effects of differentforms of theextract ofLeptadenia reticulata [Aque-

ous extract (JAE), Padavashesha kashaya (JPK) and Tarpana kashaya (JTK)] to alleviate the experimental

immunosuppression induced by the immunotoxicant chromate (VI) in vitro.

Materials and methods: Standard cell proliferation andcytotoxicity assays like MTTassay, trypanblue dye

exclusion test, neutral red dye uptake test, NBT reduction test, determination of percentage cell survival

and estimation of markers of oxidative stress were performed in the study. The study was conducted on

primary cultures of mouse splenic lymphocytes and bone marrow derived macrophages.

Results and Conclusions: Treatment with all the three forms of the extract used in the study offered pro-

tection against chromate (VI)-induced immunosuppression and the overall protective effect was found

to be superior in the case of the aqueous extract of Leptadenia reticulata (JAE). These results confirm that

Leptadenia reticulata acts as a modulator and alleviates the immunosuppressive conditions induced bychromate (VI).

2010 Elsevier Ireland Ltd. All rights reserved.

1. Introduction

Leptadenia reticulata (Retz) Wight & arn is a widely used

plant in the Indian systems of medicine. Ancient ayurvedic lit-

erature claims Leptadenia reticulata as an immune booster and

rejuvenator (Bhavamishra, 2004) and it is prescribed to increase

longevity (Agnivesa, 2001). The plant popularly known as Jee-

vanti in the Indian systems of medicine is a perennial leafy shrub,

which has been reported to possess several medicinal proper-

ties (Anjaria et al., 1975; Nadkarni, 1982; Sathiyanarayanan et al.,2007).

Chromate (VI) is a widely employed industrial chemical. Both

trivalent and hexavalent forms of chromium suppress the immune

response with hexavalent chromium being more suppressive than

trivalent chromium (Arunkumar et al., 2000).

The objective of the present investigation is to study the

effects of aqueous extract and kashayas of Leptadenia reticu-

lata extract on mouse splenic lymphocytes and bone marrow

derived macrophages with respect to cell survival, cell prolifer-

Corresponding author. Tel.: +91 80 22961461.

E-mail address: [email protected](M. Sreepriya).

ation, cytotoxicity and anti-oxidant status during experimental

immunosuppression induced by chromate (VI).

2. Materials and methods

2.1. Collection of plant material and preparation of extracts

Aerial parts of taxonomically identified Leptadenia reticulata

(Retz) Wight & arn was collected from Dhanwantri vana, Min-istry of Forests, Government of Karnataka, JB campus, Bangalore,

Karnataka. The plant material was authenticated by Dr. K. Ravi

Kumar, Senior Botanist, Foundation for Revitalisation of Local

Health Tradition (FRLHT) Bangalore, India, where a voucher spec-

imen (accession numbers 008785 and 008786) of the plant was

deposited in the herbarium. The plant material was shade dried

and coarsely powdered. The coarse powder was then utilised for

the preparation of the aqueous extract by soxhlation technique.

The percentage yield of the extract was found to be approximately

26%.

Kashayas from Jeevanti were prepared according to classi-

cal procedure from ayurvedic literature Sharangadhara Samhita

(Acharya Sharanghadhara, 2000). Following these procedures

Padavashesha Kashaya (JPK) and Tarpana Kashaya (JTK) were pre-

0378-8741/$ see front matter 2010 Elsevier Ireland Ltd. All rights reserved.

doi:10.1016/j.jep.2010.06.043


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506 V. Girishkumar et al. / Journal of Ethnopharmacology 131 (2010) 505508

pared. All the preparations (JAE, JPK and JTK) were freeze dried and

stored at 20 C.

2.2. Animals

C57 BL/6J mice were used for the isolation of splenic lympho-cytes and bone marrow derived macrophages. The animals were

procured from authentic animal source of the university and all

the experiments with animals were strictly in adherence with the

guidelines specified by the CPCSEA, India.

2.3. Chemicals

Sterile readyto use DulbeccosmodifiedEaglesmedium (DMEM

AL-068), penicillin, streptomycin, kanamycin sulphate and ampho-

tericin B were procured from Hi-media, Mumbai India. Foetal

Bovine Serum (FBS) wasprocuredfrom GIBCO,GRL USA. MTT, Nitro

Blue Tetrazolium chloride (NBT), thiobarbituric acid and neutral

red were procured from Sigma chemical company, St. Louis, MO,USA. All other chemicals used were of analytical grade and were

purchased from local companies.

2.3.1. Isolation of mouse splenic lymphocytes and bone marrow

derived macrophages

The mice were sacrificed by cervical dislocation under ether

anaesthesia following animal ethics guidelines. Spleen was asep-

tically removed and then the lymphocytes were isolated from

the spleen under aseptic conditions as described by Sai Ram et

al. (1997). Lymphocyte preparations having >90% viability (as

assessed by trypan blue dye exclusion test) was suspended in com-

plete DMEM containing 10% FBS supplemented with antibiotics

(penicillin100 IU/ml, streptomycin 100g/ml,kanamycin sulphate

20g/ml) and fungizone (amphotericin B 20g/ml). The cell count

was adjusted to contain 5106 cells/ml and plated onto 96-well

micro-titre plates (Tarsons,India). For theisolationof macrophages,

bone marrow suspension was prepared following the method of

Botran and Vetvicka (1995) and macrophages isolated from the

bone marrow suspension following the method of Fortier and Falk

(2001). Macrophagepreparations having>90% viabilitywere taken.

The cell count was adjusted to 1106 cells/ml and the cells were

plated onto 96-well micro-titre tissue culture plates.

2.3.2. Experimental regimen and assays

After plating the cells Leptadenia reticulata extracts (JAE, JPK

and JTK) were added to the wells at different concentrations, rang-

ing from 1g/ml to 1000g/ml. After pre-incubation of the cells

with varying concentrations of the extract for 30 min, cells were

challenged with potassium dichromate (10g/ml) for the induc-

tion of immunosuppressive conditions and oxidative stress. Mediacontrol wells containing DMEM alone and cell control wells con-

taining cells + media were put up for comparison purpose. The

plateswereincubated at37 C,5%CO2 and95%humidityin a carbon

dioxide incubator (Forma scientific, USA) for 72h. Splenic lympho-

cytes were used for MTT assay (Mosmann, 1983), trypan blue dye

exclusion test (Vasiliev et al., 2002) and lipid peroxide estimation

(Ohkawa et al., 1979), whereas bone marrow derived macrophages

were employed for the MTT assay, NBT reduction test (Williams

et al., 1977), neutral red dye uptake test (Parish and Mullbacher,

1983) and estimation of Nitric oxide release (Green et al., 1982).

2.4. Statistical analysis

All the experiments were carried out in triplicate on two differ-ent occasionsand the statistical analysis of the datawas determined

by Students t-test.

3. Results

3.1. Effect of Leptadenia reticulata extracts on cell proliferation

(MTT assay), cell viability (trypan blue dye exclusion test) and

levels of lipid peroxides

Table 1 shows the results of MTT assay (lymphocytes and

macrophages), trypan blue dye exclusion test and levels of lipid

peroxides on mouse splenic lymphocytes. Results indicate that

the cells treated with chromate (VI) showed marked suppression

of cell proliferation, decreased cell viability, and increased levelsof lipid peroxides which was found to be statistically significant

(P< 0.001)as compared to control. It wasobservedthat allthe three

different forms of the extract were able to protect against chro-

mate (VI)-induced immunotoxicity on splenic lymphocytes with

respect to cell proliferation and cytotoxicity which was found to

be statistically significant. On the contrary, out of the three types

of extracts used in the study only JAE was able to significantly

(P< 0.01) protect against lipid per oxidation induced by chromate.

Although treatment with JPK and JTK showed decrease in the lev-

Table 1

Effects ofLeptadenia reticulata extract (JAE, JPK, JTK dose 3.125g/ml) on cell proliferation and survival (MTT assay), cell viability (TBDE) and levels of lipid peroxides on

mouse splenic lymphocytes during chromate-induced immunosuppression.

Sl. No. Groups Cell proliferati on (as absorbance

a

) and cell survival (as %) by MTT assay Cell viability

b

(TBDE) LPO

c

Lymphocytes Macrophages

Cell proliferation % Cell proliferation %

1 Control 1.037 0.017 100 1.069 0.021 100 1.320 0.08 0.200 0.018

2 Chromate 0.872 0.041*** 84 0.892 0.050*** 83 0.585 0.05*** 0.251 0.021***

3 JAE per se 0.991 0.001*** 96 1.050 0.022*** 98 1.230 0.05*** 0.199 0.005***

4 JAE + Cr 0.910 0.008* 88 0.976 0.017** 91 0.825 0.04*** 0.214 0.008**

5 JPK per se 1.005 0.019 *** 97 1.083 0.041*** 101 1.365 0.05*** 0.207 0.003***

6 JPK + Cr 0.950 0.053* 92 0.981 0.059* 91 1.020 0.03*** 0.239 0.00NS

7 JTK per se 1.040 0.070*** 100 1.022 0.051** 96 1.125 0.04*** 0.191 0.010***

8 JTK + Cr 0.994 0.068** 96 0.963 0.047* 90 0.750 0.06*** 0.264 0.02NS

Statistical analysis by Students t-test: values are expressed as meanSD. All the groups were compared against the chromate treated immunosuppressed group (Sl. No. 2).

NS: non significant. Cell survival was expressed vs. control group (considered as 100%).a Absorbance at 570nm MTT assay.b Cell count in lakhs/ml trypan blue dye exclusion test (TBDE).c

n moles of MDA/mg protein lipid peroxides (LPO).* P< 0.05.** P< 0.01.

*** P< 0.001.


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V. Girishkumar et al. / Journal of Ethnopharmacology 131 (2010) 505508 507

Table 2

Effects of Leptadenia reticulata extract (JAE, JPK, JTK dose 3.125g/ml) on neutral red dye uptake, NBT reduction and nitric oxide release during chromate VI-induced

immunosuppression on mouse bone marrow derived macrophages.

Sl. No. Groups Neutral red dye uptake (OD at 570 nm) NBT reduction (OD at 570 nm) Nitric oxide release (mol)

1 Control 0.134 0.027 0.115 0.003 15 0.215

2 Chromate 0.074 0.002*** 0.103 0.002*** 31 0.675***

3 JAE per se 0.168 0.044*** 0.158 0.012*** 18 1.700***4 JAE + Cr 0.151 0.030*** 0.119 0.004*** 31 0.89NS

5 JPK per se 0.140 0.019*** 0.111 0.009NS 18 2.040***

6 JPK + Cr 0.128 0.016*** 0.110 0.009* 30.4 0.980NS

7 JTK per se 0.147 0.021*** 0.112 0.00NS 19.56 4.16***

8 JTK + Cr 0.122 0.006*** 0.111 0.008* 32.88 3.43NS

Statistical analysis by Students t-test: values are expressed as meanSD. All the groups were compared against the chromate treated immunosuppressed group (2). NS:

non significant.* P< 0.05.

*** P< 0.001.

els of lipid peroxides the results were found to be statistically

non significant. The dosage of 3.125g/ml (fixed after the pilot

study based on MTT assay) was found to give the best results

and hence was fixed up as the optimal dosage for all other spe-

cialised studies. The results of MTT assay on macrophages werein accordance with the results obtained with splenic lympho-

cytes.

3.2. Effect of Leptadenia reticulata extracts on mouse bone

marrow derived macrophages (neutral red dye uptake test, NBT

reduction test and nitric oxide release)

Table 2 shows the effect of different forms of Leptadenia reticu-

lata (JAE, JPK and JTK) on neutral red dye uptake, NBT reduction

and nitric oxide release on bone marrow derived macrophages.

Immunotoxicity induced by chromate (VI) resulted in statisti-

cally significant decrease in the neutral red dye uptake, NBT

reduction, and an increase in nitric oxide release ( P< 0.001) as

compared to control. Pre-treatment of the cells with JAE, JPKand JTK prior to chromate (VI) challenge resulted in statisti-

cally significant increased uptake of the neutral red dye and

increased NBT reduction whereas the results of nitric oxide release

were found to be statistically non significant with all the three

extracts.

4. Discussion

Activation of nuclear factor kappa B and the resultant down

regulation of the anti-apoptotic genes CIAp1 and CIAp2 following

chromate treatment culminates in apoptosis (Chen et al., 2002).

This could be the reason for the decreased cell proliferation and

increased cytotoxicity observed in chromate (VI) treated group. It

is believed that the protective effects of JAE, JPK, and JTK against

chromate (VI)-induced toxicity could be attributed to the pres-ence of triterpenoids like alpha and beta amyrin and beta amyrin

acetate reported to be present in Leptadenia reticulata (Noor et al.,

1992). These compounds are reported to exert strong inhibitory

effect on the activation of NF-B and CREB (cyclic AMP response

element-binding protein) (Vitor et al., 2009). Hence it is possible

that Leptadenia reticulata extract suppressed NF-B activation and

thereby prevented chromate (VI)-induced inhibition of cell prolif-

eration.

The increasedlevelsof lipid peroxidesobserved in thechromate

(VI) treated cells indicate the severity of oxidative stress resulting

from chromate (VI) exposure. A significant reduction in the levels

of lipid peroxides in the group of cells that were treated with JAE

prior to chromate (VI)challengeindicates theanti-oxidantand anti-

peroxidative effects of JAE extract. The anti-peroxidative effectsof JAE could be due to the presence of triterpenes like amyrin,

simiarenol in Leptadenia reticulata which have been reported to

replenish the GSH levels and thereby act as a very efficient detoxi-

cant and free radical scavenger (Oliveira et al., 2005).

In the present investigation cells treated with chromate (VI)

showed signs of oxidative stress as evidenced by a significant

increase in the levels of NO as compared to control. This is in accor-dance with earlier reports by Devi et al. (2004) which showed an

increase in the levels of nitric oxide in the macrophages conse-

quent to chromate (VI) exposure. All the three forms of the extract

used in thecurrent study were found to be ineffective in preventing

the increase in nitric oxide release induced by chromate (VI). This

implicates that the dosage employed in the present study may not

be sufficient enough to suppress the production of NO and a higher

dose might be effective in minimizing the deleterious production

of NO significantly.

To summarize, Leptadenia reticulata has immunomodulatory

activity and at a dosage of 3.125 g/ml, all the three forms of

Leptadenia reticulata extract used in the study offered protection

against experimental immunosuppression induced by chromate

(VI) with respect to cell proliferation, survival and prevention of

cytotoxicity. On the contrary considering the anti-oxidant activity,

only JAE was found to exhibit protection against lipid peroxidation

whereas none of the extracts was found to protect against nitric

oxide release induced by chromate.It is believed that thedifference

in activity between the extracts could be attributed to the differ-

ence in concentration of active pharmacological ingredient (API)

responsible for protection against chromate (VI)-induced immuno-

suppression. The overall degree of protection observed against

chromate (VI) toxicity was in the order JAE> JPK > JTK. Detailed in

vivo studies are required to have a better understanding of the

mechanisms involved in the immunomodulation.

Acknowledgements

The authors are thankful to Prof. S.K. Sarangi, Chairman,Depart-

ment of Microbiology and Biotechnology, Bangalore University,

Bangalore for providing the necessary infrastructural facilities

required for the work, and Prof. K.S. Jayashree, Former Head,

Department of PG studies in Dravyaguna, Government Ayurveda

Medical College, Bangalore, for initiating the idea behind this work.

References

Acharya Sharanghadhara, 2000. Kvatha kalpana. In: Shastri, P.P. (Ed.), Sharanghad-hara Samhitha. Panduranga Jawaji Publications, Bombay, pp. 144145.

Agnivesa, 2001. Shadvirechanashataashriteeya adhyaya. In: Acharya, J. (Ed.),Charaka Samhita. Chaukambha Sanskrit Sansthan, Varanasi, pp. 3234.

Anjaria, J.V., Varia, M.R., Janakiraman, K., Gulati, O.D., 1975. Studies on Leptadeniareticulata: lactogenic effects on rats. Indian Journal of Experimental Biology 13,

448449.Arunkumar, R.I., Rajasekaran,P., Michael,R.D., 2000.Differential effect of chromiumcompounds on the immune responseof the African mouth breeder Oreochromismossambicus (Peters). Fish and Shellfish Immunology 10, 667676.


5/5


508 V. Girishkumar et al. / Journal of Ethnopharmacology 131 (2010) 505508

Bhavamishra, 2004. Gudoochyadi varga. In: Pandey, G.S., Chunekar, K.C. (Eds.),Bhavaprakasa Nighantu.ChaukambhaSanskrit Sansthan,Varanasi, pp. 295296.

Botran, R.F., Vetvicka, V., 1995. Methods in Cellular Immunology, 1st ed. CRC Press,New York.

Chen, F., Bower, J., Leonard, S.S., Ding, M., Lu, Y., Rojanasakul, Y., Kung, H.F., Vally-athan, V., Castranova, V., Shi, X., 2002. Protective roles of NF-kappa B forchromium (VI)-induced cytotoxicity is revealed by expression of I kappa Bkinase-beta mutant. Journal of Biological Chemistry 277, 33423349.

Devi, K.P., Sairam, M., Sreepriya, M., Devaki, M., Ilavazhagan, G., Selvamurthy, W.,2004. Immunomodulatory effects ofPremnatomentosa(L. Verbenaceae)extractin J 779 macrophage cell cultures under chromate (VI)-induced immunosup-pression. TheJournalof Alternative and Complementary Medicine 10, 535539.

Fortier, A.H., Falk, A., 2001. Chapter 14 1 Isolation of murine macrophages. In: Col-igan, J.E., Bierer, B.E., Margulies, D.H., Shevach, E.M., Strober, W. (Eds.), CurrentProtocols in Immunology. John Wiley and Sons, New York, pp. 18.

Green, L.C.,Wagner, D.A., Glogowski,J., Skipper,P.L., Wishnok,J.S.,Tannenbaum, S.R.,1982. Analysisof nitrate, nitrite and [15N] nitrate in biologicalfluids. AnalyticalBiochemistry 126, 131138.

Mosmann, T., 1983. Rapid colorimetric assay for cellular growth and survival:application to proliferation and cytotoxicity assays. Journal of ImmunologicalMethods 65, 5563.

Nadkarni, K.M., 1982. Leptadenia reticulata. Indian Materia Medica, vol.1., 3rd ed.Popular Prakashan Pvt. Ltd., Mumbai, pp. 738.

Noor, F., Ahmed, A.,Imtiazuddin, S.M., Khan, B.,1992.A triterpenoid fromLeptadeniapyrotechnica. Phytochemistry 32, 211212.

Ohkawa, H., Ohishi, N., Yagi, K., 1979. Assay for lipid peroxides in animal tissues bythiobarbituric acid reaction. Analytical Biochemistry 95, 351358.

Oliveira, F.A., Chaves, M.H., Almeida, F.R., Lima Jr., R.C., Silva, R.M., Maia, J.L., Brito,G.A., Santos, F.A., Rao, V.S., 2005. Protective effect of alpha- and beta-amyrin, atriterpene mixture from Protium heptaphyllum (Aubl.) March. trunk woodresin,against acetaminophen-induced liver injury in mice. Journal of Ethnopharma-cology 98, 103108.

Parish,C.R,Mullbacher,A.,1983.AutomatedcolorimetricassayforT cellcytotoxicity.

Journal of Immunological Methods 58, 225237.Sai Ram, M., Sharma, S.K., Ilavazhagan, G., Kumar, D., Selvamurthy, W., 1997.

Immunomodulatoryeffects ofNIM 76,a volatilefraction fromneemoil. Journalof Ethnopharmacology 55, 133139.

Sathiyanarayanan, L, Sinnathambi, A., Chidambaranathan, N., 2007. Anti-carcinogenic activity ofLeptadenia reticulata against Daltonsascitic lymphoma.Iranian Journal of Pharmacology and Therapeutics 6, 133135.

Vasiliev,A.V., Kiseliov,I.V., Ivanov,A.A., Feddorov,D.N., Smirnov,S.V., Terskikh,V.V.,2002. Preservation of human skin: viability criteria. Annals of Burns and FireDisasters 15, 19.

Vitor, C.E., Figueiredo, C.P., Hara, D.B., Bento, A.F., Mazzuco, T.L., Calixto, J.B., 2009.Therapeutic action and underlying mechanisms of a combination of two penta-cyclic triterpenes, alpha- and beta-amyrin, in a mouse model of colitis. British

Journal of Pharmacology 157, 10341044.Williams, G.M., Bermudez, E., Scaramuzzino, D., 1977. Rat hepatocyte primary cell

cultures.III.Improved dissociationand attachment techniques andthe enhance-ment of survival by culture medium. In Vitro 13, 809817.

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