Indian Journal of Experimental Biology Vol. 37, January 1999, pp. 27-3\

Effect of "Rasayanas", a herbal drug preparation on immune responses and its significance in cancer treatment

V Praveen Kumar, R.Kuttan* & G. Kuttan

Amala Cancer Research Centre, Amala Nagar, Thrissur 680 553, India

Received 26 August 1997; revised * October 1998

Rasayanas are considered to be immunostimulating preparations used extensively in indigenous medical practice. However there are only very few reports to substantiate this claim, and this paper gives preliminary evidence for the potentiation of immunity by Rasayanas given to mice orally. Administra­tion of Rasayanas were found to enhance the proliferation of spleen cells significantly especially in the presence of mitogen. Similar result was also seen with bone marrow cells; however mitogenic stimula­tion could not be observed. Esterase activity was found to be enhanced in bone marrow cells indicating increased maturation of cells of Iymphqid linkage. Rasayanas also enhanced humoral immune response as seen from the increased number of antibody forming cells and circulating antibody titre . These results indicate the usefulness of Rasayana as immunostimulating agent.

In recent years, there is an increasing interest in the search for potential drugs, especially, of plant origin, that are capable of modifying Immune responses (immunomodulators) with comparatively low side effects l

-5

. These immunorestorative drugs may reduce myelosuppression induced by cytoreductive therapy as seen in cancer6. Eventhough several natural and synthetic compounds were developed during the past several years, clinical value of most of the immunotherapeutic regimens tested so far has not yet been proven unequivocall/ . Rasayanas are a group of non-toxic herbal drug preparations which are used to improve the genaral health in normal and disease conditions by the stimulation of the immune system8

• Our initial studies have indicated that some of the Rasayanas could increase the total leukocyte counts and percentage of neutrophils in the peripheral blood9

.

In additiotT, Rasayana treatment increased the life span of ascites tumour-bearing mice and reduced the solid tumour volume induced by Oaltons lymphoma ascites (OLA) cells9

. Further, Rasayanas significantly protected the mice from cyclophosphamide1o and radiation ll induced myelosuppression and subsequent leukopenia. In the present study we have evaluated the effect on

• Correspondent author

T -, B- and bone-marrow cells 10 mIce treated with Rasayanas.

Materials and Methods

Tissue culture media RPMI- 1640 and OMEM were purchased from Himedia laboratories, Bombay. Phytohemagglutinin (PHA) was obtained from Oifco, USA and concanavalin-A (CON A) from Sigma St.Louis, USA. Pararosanil ine and alpha-naphthyl acetate were obtained from Loba Chern ie, Bombay. Haematoxylin was purchased from E.Merck (India). 3H-thymidine was purchased from BRIT, Babha Atomic Research Centre, Bombay. All other reagents were of Analytical Reagent quality.

Balb/c mice (4-6 weeks old) were used for all the studies. They were obtained from National Institute of Nutrition, Hyderabad and maintained in air controlled rooms. The animals were fed with normal mouse chow (Lipton, India) and water ad libitum.

Brahma Rasayana (BR), NaraSimha Rasayana (NR), Ashwagandha Rasayana (AR) and Amruthaprasham (AP) were purchased from "Vaidyaratnam Oushadha sala", Thrissur. The main ingredients were described elsewhere9

.

Rasayana (50/mg/animaVdose) were given orally on five alternate days before all the

28 INDIAN J EXP BIOL, JANUARY 1999

experiments. This dosage has been found earlier to improve WBC counts in normal mice9

.

Assay of lymphocyte blastogenesis- Whole spleen cells without separating macrophages were used to measure lymphocyte blastogenesis animals were sacrificed 24 hI after the completion of the drug treatment, spleens were removed and made into s ingle cell suspension by passing through a wire mesh. The cells from treated and untreated animals were cultured (I06cells/ml) in presence and absence of mitogens such as PHA (3 and 61lg/ml ) and CON A (5 and 101lg/m l) in RPMI-1640 medium containing 10% FCS (fi na l volume 3 ml) and ant ibiotics in a humidified atmosphere of 5% CO2 at 37°C. After 48 hr, 3H_ thymidine was added

(2IlC i/via l) and incubation continued for 18hr Further DNA was precipitated using perchioric ac id and incorporated radioactivi ty was counted us ing liqu id sc inti llation counter l2

.

Bone-marrow cell proliferation assay-Total bone marrow cells without separation were used to determine the effect of Rasayanas on bone marrow ce ll proliferat ion. The procedure employed was the same as above. Instead of spleen cells bone­marrow ce ll s ( 106 cel ls/ml) from treated and non­treated animals were cul tured in presence and absence of mi togens (P HA-61lg/ml and Con A 5 Ilg/ml) . The prol iferation of bone-marrow cells were assesed from the amount of radioactive thymidine incorporated in to the DNA .

Alpha-naphthyl acetate esterase activity in bone­marrow cells-Naphathyl acetate esterase activity in the bone marrow is an indicator of maturation of stem ce ll s to monocytes-macrophages. Total bone marrow ce ll s from the animals treated with and withoui Rasayanas were made into a single cell suspens ion. A .smear was prepared, dried and stained accord ing to the method of Bancroft and

Cook l3. Briefly, the slides were incubated in a

mixure (PH 6.1) containing 44.5 ml phosphate buffe:r (PH 7.6), hexazotized pararosaniline (1 .2 ml 5% pararosaniline and 1.2 ml 4% NaN02) and a lpha-naphthyl acetate (50mg in 2 .5 ml ethylene glycol monoethyl ether) for 45 min at room temparature. It was washed and counter stained with haematoxylin . A total of 4000 cells were counted in trip licates and number of cells positive for esterase were counted .

Anlibody forming cells-It was done by Jern 's plaque assay using a modified slide techniq ue. 14

Along with the last dose of drug treatment a ll animals received SRBC (2 .5x I 06 ce lls) intraperitoneally. The animals were sacrificed on various days and spleens were processed into s ingle ce ll suspension (8x I 06 cells/ml) in HBSS. To 0.5 m1 of 5% agarose prepared in HBSS, 50111 of 7% SRBC and 50111 of spleen cell suspension were added, mixed we ll and poured over a glass slide.The slides were allowed to solidi fy and incubated wi th fresh rabbit serum as a source of complement for I h at 37°C. The plaques formed were counted us ing a colony counter and represented as plaque forming cells (P FCs)/million spleen cells.

Circulating antibody litre-A long with the last dose of Rasayana treatment a ll the animals received 0.1 ml of 20% SRBC intra peri tonea lly. Blood was collected before and on various days after the inj ect ion of SRBC from the ta il ve in . Sera samples of each group were pooled and heat inacti vated at 56°C for 30 min. Two fo ld dilutions of sera samples were made in PBS '(pH 7.2) in microtitre plates and mixed (I : I )with 1% trypsinized suspension of SRBC in PBS. Agglutination was noted after incubation for 3 -hr at room temparature l8

.

Table I- Effect of Rasayanas on Iymphocyles blastogenesis [Values are mean of ± SD of 6 different estimations)

Treatment No PHA eDNA

mitogens 6fJg/mi 3 [Jg/ !Id IOpg/m l 5fJglmi

No Treatment I 595±377 600 1±1212 2345±3 12 7421±274 5174±577

BR 1651±120 12594±776* 11 345±127* 12861±812* 11 775±298*

NR I 579±584 12386±261* 111 57± 1390* 13280± 11 68* 10299±3 18*

AR 1745±22 669 1±1191 4202±665 6856±480 3622±998

AP 1628± 150 35763±3 120* 34959± 1330* 34581±409* 3177 1±449*

*P<O.OO I

PRA VEEN KUMAR et al.: EFFECf OF RASA Y ANAS IN CANCER TREATMENT 29

Statistical analysis-Statistical analysis were done by Student's t test.

Results Effect of Rasayanas on lymphocyte

blastogenesis-The effect of various Rasayanas on the spleenic lymphocyte blastogenesis is shown in Table 1. There was a 2-fold increase in the thymidine uptake by spleen cells of mice stimulated with PHA and Con A after treatment with BR and NR. AP treated mice showed a 6-fold increase in the proliferation after PHA and Con A stimulation compared to controls. The treatment with AR did not have any effect under the same conditions.

Effect of Rasayanas on bone-marrow cell proliferation-There was a significant increase in the proliferation of bone-marrow cells of mice treated with Rasayanas (Table 2). Bone-marrow cells from BR and AR treated mice proliferated between nine to ten times hi gher than that of bone­marrow cells from the untreated control animals

Tab le 2-Effect of bone marrow proliferation [values are mean of ± SO of6 different estimations]

Treatment No mitogens PI-IA 6 I-lg/ml CON A I Ol-lg/ml No treatment 909±l54 934± l14 869± 48 AR II 115± 54 * II 528±5 12* 9630±511* NR 1023± 150 l192±219 756± 163 AR 9580±753* 97l5±52 1* 9561 ± 11 09* AP 8 19± 11 6 778± 23 707± 32 ·P<O.OOI

Tab le 3-Rasayanas on esterase acti viti y in bone-marrow cells

[V al ues are mean of three d ifferent esti mations]

T reatment Cell s wi th positve % Increase sta in ing

No treatment 684/4000 Brahma Rasayana 776/4000 20 Narasi mha 860/4000 33 Rasayana Ashwagandha 884/4000 36 Rasayana Amruthaprasham 698/4000 7.4

indicating that administration of BR and AR increases bone marrow proliferation in vivo. As the bone marrow cells did not have any mature T cell s presence of mitogens did not alter the proliferative. rate of these cells. Treatment with NR and AP did not have any effect on the proliferation of the bone marrow cells of mice.

Effect of Rasayana on alpha naphthyl acetate esterase activity-Effect of Rasayanas on esterase activity which is an indicator of maturation of monocytes in bone-marrow cells are shown in Table 3. There was an increase in the number of cells with esterase activity by Rasayana treatment. Treatment with BR,AR and NR enhanced the percentage of cells positive for esterase by 20%, 33%, and 36% respectively. AP treatment had only minimal effect.

Effect of Rasayanas on antibody forming cells-There was an increase in the number of plaque formin g cells by Rasayana treatment (Table 4). The number of PFCs increased graduall y from third day onwards and max imum number of plaques were obtained on day 5 which was 2-3 times higher than that of controls. Among the four Rasayanas studied NR was found to be most effective (958 ± 8).

Effect of Rasayanas on the circulating untibody titre-Rasayana treatment enhanced the production of antibody in normal mice (Table 5) . There was an increase of (8-) 2 times) in c irculating antibody titre by the treatment with various Rasayanas . Among the four Rasayanas studied NR was found to be the most effective () :512) in the enhancement of antibody production. A maximum titre was obtained on day) 5 for AR and day 20 for BR,NR and AP.

Discussion Rasayanas are non-toxic

extracted from plants with drug preparations various biological

Table 4--Effect of various Rasayanas un pl aque formin g ce lls [Values are means±SO of three different estimati ons]

prc;'! 1 0" splcen cell ' on \ ,H'i",!, days

T reatment 3 4 5 6 7 SRBC alone l 50±5 205±8 273±6 253±3 I 245±7 BR±SRI3C 183±6 273± 14 683± 13 577± 14 555±20 NR±SRIlC 220±7 275± 12 958±8 535± l O 378±20 AR±SRBC 108±7 220±6 282±20 468±4 2 18±6 AP±SRBC 160±3 370±3 668±9 260±22 265± 12

*p < 0.00 1.

30 INDIAN J EXP BIOL, JANUARY 1999

Table 5--Effect of variousRasayanas on circulating antibody ~itre

[Values are mean of three different estimations]

Titre values on various days

Treatment 3 5 IO 15 20 25 SRBC alone 32 32 16 16 16 16 BR±SRBC 4 32 16 128 32 32 NR±SRBC 32 64 128 256 512 128

AR±SRBC 2 64 32 32 256 128

AP±SRBC 32 64 128 128 256 256

activities including immunomodulatory activityl .4.16. According to the indigenous system of medicine in India (Ayurveda) Rasayana therapy arrests aging, increases intelligence and body strength and enables to prevent diseasess. But no systematic studies were done yet to substantiate these claims. In the present study we demonstrate the immunopotentiating activity of some Rasayanas in normal mice.

T -cells contribute the major effector mechanisms in cell-mediated immunityl7. They respond to plant mitogens such as PHA and CON A by rapid blastogenesis l2 . One of the in vitro . systems to evaluate the T-cell function is its ability to get transformed and have been shown to bear correlation with in vivo parameters of cell-mediated immunity status of the individual 12. Rasayanas such as BR, NR and AP were found to stimulate the spleen cell proliferation in presence of mitogens as seen from the increased incorporation. We have also found an increase in the weight of spleen and thymus by Rasayana treatment (data not shown).

Bone-marrow cells from Rasayana (BR and AR) treated animals also showed an enhanced proliferation in vitro. Recently we have shown that both BR and AR could protect the mice from cyclophosphamide-and radiation-induced myelo­suppression 10.1 1. One of the mechanism may be by the induction of proliferation of bone-marrow stem cells either directly or indirectly stimulating the release of factors that are involved in the regulation of hemopoiesis. Number of bone marrow cells positve for non-specific esterases were found to increase after Rasayana treatment which represent the maturation of cells of monocyte/macrophage lineagels. Rasayanas were found to enhance the number of esterase positve cells that supports the above data.!t has been reported that Withania somenifera one of the components of AR,could increase the number of GM-CFU showing an increased secretion ofGM-CSF in the plasmal.

In addition to the activation of T -cells and bone­marrow cells, administration of Rasayanas could enhance the number of plaque forming cells in the spleen and antibody titre in the circulation which are the functions ofB-cells.

Rasayanas contain several plant extracts and some of them have immunomodulatory and antioxidant activities l.4.19)0. Moreover, BR has been shown to inhibit DMBA-induced sarcoma in mice21 . Recently we have shown that Rasayanas could augment the cell-mediated immune effector arms such as NK cells,K cells and macrophages in tumour-bearing animals22

. At present we do not know the exact material involved in the stimulation of the immune system. It may be by an additive effect of several components present in plants acting synergistically in the biological system.

Acknowledgement This work was partially supported by a grant

from UGC, New Delhi.

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