Research Article Available online at

P. Sachdanandam et al/JGTPS/ Volume- 5, Issue -2, April-June 2014

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P. Sachdanandam*University of Madras, Taramani Campus,

Chennai, Tamil nadu-600113, IndiaE-mail address: [email protected]

Contact no: +91-9940574529

Address for correspondence

IMMUNOMODULATORY POTENTIAL OF KALPAAMRUTHAA AN HERBAL DRUG IN EXPERIMENTAL WISTAR RATS

INTRODUCTIONThe immune system is organized in

layers with increasing specificity, designed to protect the host from invasion by various pathogens such as allergens, viruses and bacteria (Chaplin, 2010). Modulation of immune response to alleviate disease has been of interest for a long time (Bani et al., 2005). Immunomodulation may be defined as thechange in the body's immune system, cellular, humoral and non-specific defense mechanism caused by agents that activate or suppress its function. Typically, immune system is held in homeostatic balance between immune stimulation and immune suppression (Ghule et al., 2006). The development of natural herbal products that could potentially modulate the immune system provides an alternative source

of bioactive agents with medical significance (Choi et al., 2004; Kumar et al., 2011) and some medicinal plants have been shown to exert anti-inflammatory, anti-stress and anti-cancer effects by modulating the immune functions (Singh, 1986; Thatte, 1996; Gertsch et al., 2011).The search for natural immunomodulators holds a great hope for discovering effective remedies for preventing and for treating a wide range of medical conditions. Although many herbs and natural products have been shown to exert medically beneficial effects in the treatment of various diseases including cancer, studies that are focused on investigating the immunomodulatory potential of such herbs and natural products are very scarce. Traditional Indian systems of medicines like Siddha and Ayurveda have suggested means to increase the body’s natural resistance to disease. A number of Indian medicinal plants and various

The current study investigates the immunomodulating activity of Kalpaamruthaa on male Wistar albino rats. The search for natural immunomodulators holds a great hope for discovering effective remedies for preventing and treating a wide range of diseases. Kalpaamruthaa (KA), a formulation prepared in our laboratory consists of Semecarpus anacardium Linn. nut milk extract, fresh dried powder of Emblica officinalis Gaertn. fruit and Honey. KA was suspended in Olive oil and was administered orally for 10 days at doses of 50, 100 and 200 mg/kg body weight. The immunomodulating effect on specific and non-specific immunity were studied by Haemagglutinin antibody titer, plague-forming assay, delayed type hypersensitivity reaction, quantitative haemolysis of SRBC (QHS) assay, hematological and biochemical analysis. Cyclophosphamide was used as an immunosuppressant (30mg/kg/day i.p) to evaluate the level of myelosuppression. Our present study showed a significant increase in Sheep Red Blood Cells (SRBC) induced haemagglutination antibody titre when compared to control groups. An upsurge in the number of IgM producing cells was evident from the Plaque Forming assay. A significant delayed type hypersensitivity reaction by facilitating to foot pad thickness response to SRBCs in sensitized rats was also observed. It was also found that Kalpaamruthaa boosted the phagocytic activity, increased the quantitative hemolysis of SRBC and enhanced the lymphoid organ cellularity and relative organ weight in KA treated rats when compared to control rats. These alterations in the immune parameters indicates the ability of Kalpaamruthaa as a potent immunomodulator to stimulate lymphopoiesis, humoral as well as cell mediated immunity along with macrophage activity and in preventing the myelosuppression in cyclophosphamide drug treated rats .Keywords: Cyclophosphamide, Immunomodulator, Humoral immunity, DTH response,

Phagocytic index, Kalpaamruthaa

ABSTRACTKarvannan Kanchana a, Shruthy S. Kumar a, Palanivelu Shanthi b,

PanchanathamSachdanandam a*

a. Department of Medical Biochemistry, b. Department of

Pathology. Dr. ALM P-G, Institute of Basic Medical

Sciences, University of Madras, Taramani Campus, Chennai, Tamil nadu-600113, India.

Journal of Global Trends in Pharmaceutical Sciences

Available online at www.JGTPS.com

ISSN: 2230-7346Journal of Global Trends in Pharmaceutical Sciences

Volume -5, Issue-2, pp-1499-1512, April-June 2014


Research Article


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‘rasayanas’ have been claimed to possess immunomodulatory activity (Atal et al., 1986; Puri et al., 1994; Ziauddin et al., 1996; Balachandran and Panchanathan, 1998). Kalpaamruthaa, a modified Siddha formulation prepared in our laboratory, consists of Semecarpus anacardium Linn. nut milk extract (S. anacardium) (family: Anacardiceae), fresh dried powder of Emblica officinalis (EO) fruit(E. Officinalis) (family: Euphorbiaceae) and honey. KA was found to be nontoxic up to the dose level of 2,000 mg/kg (Mythilypriya et al., 2007a). In our laboratory KA has been reported for its potent antioxidant (Arulkumaran et al., 2006; Mythilypriya et al., 2007b), analgesic, antipyretic and non-ulcerogenic properties (Mythilypriya et al., 2007c), anti-inflammatory (Mythilipriya et al., 2008) and anticancer (Veena et al., 2006; Uma rani et al., 2008) properties. Semecarpus anacardium Linn. nut milk extract is a major constituent in the drug KA and has many therapeutic applications according to the Indian system of medicine. It has been reported for the presence of phenolic compounds such as biflavanones namely, semecarpuflavanone, jeediflavanone, galluflavanone, nalluflavanone, semecarpetin and anacardiflavanone (Prakash Rao et al., 1973; Premalatha et al., 2000), sterols and glycosides (Indap et al., 1986), anacardic acid (Chattopadhyaya and Khare, 1969), vitamins, linoleic, myristic, oleic, palmitic and stearic acids (Rai et al., 2000). Therapeutically it is proved to have antioxidant, antiinflammatory (Selvam et al., 2004), hypoglycaemic (Arul et al., 2004), anti-arthitic, immunomodulatory activities and anti-cancer (Ramprasath et al., 2006; Nair et al., 2009), anti-diabetic properties (Haseena et al., 2012).The next major constituent of KA is Emblica officinalis fruits. The fruits of E. Officinalis are widely consumed raw, cooked, or pickled, and are principal constituents of many Ayurvedic preparations (Scartezzini and Speroni, 2000). The Emblica fruit have been reported to contain constituents with variable biological activity such as vitamin C (Raghu et al., 2007), hydrolysable tannins (Emblicannin A, Emblicannin B, Punigluconin, Pedunculagin), Gallo ellagitannoids, flavonoid (rutin) (Ghosal et al., 1996), trigalloyl glucose (Damodaran and Nair, 1963) and phyllemblic acid (Pillay and

Iyer,1988).It has many pharmacological activities viz: Antioxidant (Scartezzini et al., 2006), Antidiabetic activity (Sabu and Kuttan, 2002), cytoprotective and immunomodulating (Sai Ram et al., 2002; Liu et al., 2012), hepatoprotective and antitumour activities (Sutapa et al., 2013). It is also used as antimicrobial (Rani and Khullar, 2004) and anti inflammatory agent (Perianayagam et al., 2004).Honey, the natural product from honeybees, is a mixture of carbohydrates like fructose, glucose and sucrose etc. It also contains minerals and proteins, with a water content of about 17.2% (White, 1979), honey also contains flavonoids at low concentrations, acquired due to the contact with pollen, which is rich in these secondary metabolites with high antioxidant activity (Campos et al., 1997; Almaraz and Campos, 2007). Combined interaction of the phytochemicals present in S. anacardium, E. Officinalis and honey may have a synergistic effect and as such Kalpaamruthaa can serve as an efficient immune stimulatory compound. The following study was carried out to determine and validate the potency of Kalpaamruthaa as an effective immunomodulator.

MATERIALS AND METHODSKalpaamruthaa

The major constituent of KA is Semicarpus anacardium Linn. nut milk extract commercially known as Serankottai nei and it was prepared according to the Formulary of Siddha Medicine (1972). Emblica fruit was air dried, powdered and the fine powder was used for the compound preparation; commercially available honey was obtained and added to the above-mentioned ingredients for the preparation of Kalpaamruthaa. The components were authenticated by Dr. S. Jayaraman, Botanist, Plant Anatomy Research Centre, Chennai, Tamilnadu.

ChemicalsPara rosaniline hydrochloride and

cyclophospamide were obtained from Loba Chemie (Mumbai,India). Haematoxylin was purchased from Glaxo (Mumbai, India). Sheep blood was collected from local slaughter house and RBC was isolated in Alsevers solution. All



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other chemicals and solvents used were of analytical reagent grade.

AnimalsMale Albino rats of Wistar strain 8–10

weeks of age weighing between 120-150g were used in this study. The animals were housed in polypropylene cages under a control environment with 12±1h light/dark cycles and maintained at a temperature between 27°C and 37 °C, and were given a commercial diet with water ad libitum. Animal experimentation was conducted according to the institutional regulations (IAEC no. 01/07/11).

Experimental Design and DosageThe animals were divided into four

groups of six each and treated accordingly. Group I (control) received olive oil. KA was suspended in Olive oil and was administered orally for 10 days at doses of 50, 100 and 200 mg/kg body weight i.e. Group II rats received KA at dosage of 50 mg/kg body weight; Group III rats received KA at dosage 100 mg/kg body weight and Group IV rats received KA at dosage 200 mg/kg body weight. The dose volume was 0.2 ml. Control animals received the same volume of olive oil for the same duration.

EXPERIMENTAL PROCEDUREAt the end of the experimental period

(10 day treatment + 5 day immunization), all the rats were sacrificed under anesthetic condition. Blood samples were collected from the caudal vein of the rat into EDTA rinsed vials for analysis of hematological parameters. Vital organs such as Liver, Spleen, thymus and kidney were removed immediately.

Haematological parameters, Body and Vital organ weights

Blood was collected from the caudal vein and parameters such as RBC count, total WBC counts (haemocytometer), differential count (Leishman’s stain) were recorded at the end of the experimental period. The animals were weighed before and after the treatment period and the weight of vital organs such as liver, spleen, thymus, and kidney were recorded and expressed as relative organ weights.

Lymphoid organ cellularity and α-esterase positive cells

Single cell suspensions were prepared in RPMI-1640 medium from bone marrow (from femur), spleen and thymus for cell count. Cell counting was done by using the Neubauer chamber (Raisuddin, 1990). A smear of the bone marrow cells from the above preparation was made on clean glass slides and stained with Para rosaniline hydrochloride and counter stained with hematoxylin to determine the non specific α esterase positive cells according to the method of Bancroft & Cook, (1984).

Liver function testsActivities of serum glutamate oxalate

transaminase (SGOT) and serum glutamate pyruvate transaminase (SGPT) were analysed by the method of King, (1965).

Assessment of humoral immune functionsAfter the treatment period of 10 days,

the animals were challenged with 0.2 ml of 10% Sheep Red Blood cells (SRBC), i.p. On the 5th day of immunization, the following parameters of humoral immunity were studied.

Haemagglutinin titre (HT) assayHT assay was performed using the

procedure of Bin-Hafeez et al., (2001). On the fifth day of immunization, blood was collected from orbital plexus of each rat for serum preparation. Serial two-fold dilution of serum was made in 50 μl of PBS (pH 7.2) in 96 well microtitre plates (Tarsons Products, Calcutta) and mixed with 50 μl of 1% SRBC suspension in PBS. After mixing, plates were kept at room temperature for 2 h. The value of antibody titre was assigned to the highest serum dilution showing visible haemagglutination.

Plaque-forming cell assayThe PFC assay was performed using the

method of Raisuddin et al., (1991). On the fifth day of immunization with SRBC the animals were sacrificed. The spleen was removed, cleaned free of extraneous tissues, and a single cell suspension of 106 cells/ml was prepared from it in RPMI-1640 medium. For PFC assay, the SRBC were prepared at a cell density of 5×108 cells/ ml in PBS. One milliliter of SRBC in medium along with 0.5 ml of diluted guinea



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pig complement (1:10 diluted with normal saline) was added to 1 ml of spleen cell suspension. Cuningham chambers were prepared using glass slide, coverslips and double sided tape. The chambers were loaded with a known volume of assay mixture, sealed with petroleum jelly and incubated at 37°C for 1 hour. The plaques were counted under a light microscope (Olympus BX50) and expressed as PFC per 106 spleen cells.

Quantitative haemolysis of SRBC (QHS) assay

QHS assay was performed using the methods of Simpson and Gozo, (1978) with some modification (Bin Hafeez et al., 2001) Spleens were removed and a cell suspension of 1×106/ml was prepared in PBS. One milliliter of 0.2% SRBC and 1 ml of 10% guinea pig serum were mixed with cell suspension and incubated for 1 h at 37 °C. After centrifugation at 3000 rpm for 3 min, optical density of the supernatant was measured at 413 nm using a spectrophotometer (Shimadzu UV- 1201).Assessment of cellular immune function

For assessment of cellular immune function, delayed type of hypersensitivity (DTH) response was evaluated in the KA treated rats.Delayed type of hypersensitivity response

The DTH response was determined using the method of Raisuddin et al., (1991). On the day of termination of the treatment, animals were immunized with 1×109 SRBC, subcutaneously. On the fifth day of immunization, all the animals were again challenged with 1×108 cells in the left hind footpad. The right footpad injected with the same volume of normal saline served as trauma control for nonspecific swelling. Increase in footpad thickness was measured 24 h after the challenge using dial caliper.

Assessment of nonspecific immune functionsFor assessment of nonspecific immune

functions, phagocytic activity of macrophages was studied. Four groups of animals were treated as described above.

Phagocytic activity of macrophagesThe phagocytic activity of peritoneal

macrophages was evaluated using the

suspension assay as described by Fujiki & Yano, (1997) with some modification.0.1 ml of aliquot of cells having density of 1×106 cells/ml were mixed with 0.1 ml RPMI-1640 medium containing 20% FCS and 1×108 cells/ml heat treated yeast (Saccharomyces cereviceae) cells. The mixture was incubated at 37ºC for 1 h with occasional shaking. After incubation, 50 μl of this mixture was smeared on the glass slide, air dried and stained with Wright–Giemsa stain. The slides were observed under light microscope using oil immersion. At least 500 cells were counted. The phagocytic activity was expressed as phagocytic index (PI).

Cyclophosphamide-induced myelosuppression

Cyclophosphamide-induced myelosuppression was done by the method proposed by Ziauddin et al., (1996). Albino rats were divided into five groups containing six rats each. Group I served as normal control received olive oil. Group II was administered with only cyclophosphamide at the dose of 30 mg/kg, i.p. while groups III, IV and V rats received cyclophosphamide with varied concentrations of Kalpaamruthaa (50–200mg/kg, p.o.) orally for 10 days. On day 11, blood samples were collected from the retro-orbital plexus of individual animals and analyzed for haematological and serological parameters.Statistical Analysis

The values are expressed as mean±SD for six rats in each group. Significant difference between the mean values were determined by one-way analysis of variance (ANOVA) followed by the Tukey’s test for multiple comparison using Statistical Package for Social Sciences (SPSS) computer package. Significant difference between control and treatment groups were assigned at p<0.001, p<0.01 and p<0.05.

RESULTSEffect of Kalpaamruthaa on Body weight, relative organ weight and haematological

parametersNo toxicity or mortality was observed in

the KA-treated animals with varied doses. Significant body weight differences were not observed among the different groups of animals (data not shown). At the dose of 100 and 200 mg/kg, a significant increase (p<0.05), (p<0.01), in relative weight of spleen and liver



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was observed when compared with control (Table 1). This increase in liver weight did not reflect any abnormality as assessed by LFT.RBC count, WBC count, differential count were significantly increased in rats treated with KA at a dose of 100 mg /kg (p<0.05) and 200 mg/kg (p<0.01) body weight when compared with control animals receiving olive oil alone (group I) (Table 2). KA did not alter the relative organ weights of kidney and thymus.

Effect of Kalpaamruthaa extract on liver marker enzymes

There was no significant elevation in the levels of SGOT and SGPT as a result of treatment with KA at any of the doses used in this study (data not shown).

Effect of Kalpaamruthaa on Lymphoid organ cellularity and α-esterase positive cells

The spleen and thymus cellularity significantly (p<0.01) increased at the dose of 200 mg/kg when compared with control animals (group I) (Table 3). In the case of bone marrow, animals receiving KA at the dose of 100 and 200 mg/kg showed a significant (p< 0.05), (p<0.01) increase in cellularity when compared with control rats. Moreover the numbers of α-esterase positive cells were also found to be increasing significantly at the dosage of 100 and 200 mg/kg b.wt. (p< 0.05), (p<0.01), in the KA treated groups compared to controls.

Effect of Kalpaamruthaa on humoral immunity

Effect of KA on humoral immunity parameters viz., PFC assay and QHS assay, are shown in Figure 1 & 2, respectively. Kalpaamruthaa showed an elevated response at a dose of 100 and 200 mg/ kg, but at doses of 50 mg/kg, no effect was observed when the data’s were compared with vehicle-treated control animals (group I).

In haemmaglutination titre (Table 4), doses of 50, 100 and 200 mg/kg showed titre values of 6.43, 7.64, 10.51 respectively, while the titre value of controls (group I) was 6.09. Thus showing increase in HA titer in all the treated groups on comparison to control group.

Effect of Kalpaamruthaa on cell-mediated immune parameters

Kalpaamruthaa at the doses of 50, 100 and 200 mg/kg elicited a significant increase in DTH response which is peaking in Group IV (p<0.01) (Table 5) when compared to control animals (group I).Heightened DTH response suggests activation of cellular immune system.

Effect of Kalpaamruthaa on phagocytic activity of macrophages

The results of effect of KA on macrophage functions are presented in Figure 3. KA at the dose of 100 mg/kg body weight (p< 0.05) and 200 mg/kg body weight (p<0.01) boosted the rate of phagocytic activity of peritoneal macrophage as evident from the increase in phagocytic index when compared with control animals (group I).There was no significant effect at the dose of 50mg/kg body weight.

Effect of Kalpaamruthaa on Cyclophosphamide-induced

myelosuppressionCyclophosphamide at the dose of 30

mg/kg, i.p. caused a significant reduction in the RBCs, WBCs and Hb level. Treatment of cyclophosphamide receiving group with Kalpaamruthaa (50–200 mg/kg) resulted in a restoration of bone marrow activity. Significant reduction in white blood cell count was observed in animals treated with cyclophosphamide alone (group II) as compared to the control group (group I). Kalpaamruthaa significantly increased the levels of WBC in adose-dependent manner as compared to the cyclophosphamide receiving group.(Table 6)DISCUSSION

The immune system is a complex and highly developed system, yet its mission is simple: to seek and kill invaders. A defectiveimmune system is found to be involved in the development of several complex chronic diseases such as cancer (Bultz and Carlson, 2005), cardiovascular diseases (Busse et al., 2001) and diabetes (Sepa et al., 2005). There is no doubt that the relationship between the immune system and human health is very intimate. Immunomodulation through natural substances may be considered as an alternative for the prevention and cure of disease. Plant products can serve as an excellent class of immunomodulators, as a variety of plant derivatives such as polysaccharides, lectins,



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peptides, flavonoids and tannins have been reported to modulate the immune system in various in vivo models (Shivaprasad et al., 2006). Hence, many researches are now focusing on discovering natural products that possess the potential to strengthen all aspects of the immune system (Tapsell et al., 2006; Hashemi and Davoodi, 2012).

The results obtained in the present study indicate that Kalpaamruthaa, an indigenous modified Siddha formulation prepared in our laboratory is one among the most promising candidate having immunomodulatory effect by stimulating the immune system. Kalpaamruthaa showed an overall stimulatory effect on the humoral immunity, cellular immunity and non-specific immunity in rats. Stimulatory effects were mostly observed at the dosage of 100 mg and 200mg/kg when compared with control group. Innate immunity largely depends on the activity of granulocytes and macrophages, while adaptive immune response depends upon lymphocytes, which provide long term immunity (Raphael & Kuttan, 2000). The concept of immunomodulation relates to nonspecific activation of the function and efficacy of macrophages, granulocytes, complement, NK cells and lymphocytes and also to the production of various effector molecules generated by activated cells (Jayathirtha and Mishra, 2004). KA at doses of 100 and 200 mg/kg was found to increase RBC count, total WBC count, lymphocyte distribution and lymphoid organ cellularity significantly indicating that the Kalpaamruthaa could stimulate the haemolymphopoeitic system. A similar effect of S. anacardium and E. officinalis has been observed in previous studies (Ramprasath et al., 2005, Haque et al., 2001; Suja et al, 2009). The increment in the number of bone marrow cells and differentiating stem cells with alpha-esterase activity in Kalpaamruthaa treated animals also shows the effect of it on enhancing the immunological response. The spleen weight along with its cellularity was also found to be drastically increased. This may be partly due to augmenting effect of KA on the lymphocytes and bone marrow haematopoietic cells, which ultimately reside in spleen. However, this residence may be temporary and in due course of time normalcy may ensue. Even though,

there was an increase in liver weight, estimation of the LFT enzymes did not reflect any toxicity. Mythilipriya et al., 2007a have already shown in sub acute toxicity study of KA that it did not alter GOT, GPT and alkaline phosphates (ACP) levels either in serum or liver in rats up to 30 days.

The main phytochemical constituents of Kalpaamruthaa are flavonoids, ascorbic acid, polyphenols, tannins, sugars, sterols, glycosides etc (Mythilipriya et al., 2007b). It can be assumed that some of the constituents might be having mitogenic effects, which in turn lead to stimulatory effect in the immunocompetent cells. Some of these constituents also possess antioxidant properties and they might have further added to the immunostimulatory potential, because a number of antioxidants have been reported to possess immunomodulating properties (de La Fuente and Victor 2000; Devasagayam and Sainis, 2002). Combined interaction of these phytochemicals present in S. anacardium and E. officinalis improved the effectiveness of our formulation in several ways.

Most organisms survive through innate immune mechanisms alone, but vertebrates have evolved an additional system for pathogen recognition and elimination that consists of T and B lymphocytes and humoral factors known as the adaptive immune system (Calder, 2006). The T helper type 1 (Th1) lymphocytes secrete cytokines such as interferon and interleukin-2 and stimulate type 1 (cellular mediated) immunity characterized by intense phagocytic activity by cytotoxic T lymphocytes and other myeloid cells, which regulate the synthesis of antibodies as well as direct killer cell activity and the inflammatory response of delayed type hypersensitivity (Radoja et al., 2006). Conversely, Th2 cells secrete different kinds of signaling proteins such as interleukins 4, 5, 9 that stimulate type 2 (humoral mediated) immunity characterized by high antibody production by B lymphocytes (Sproul et al., 2000). These antibodies can neutralize microorganisms or toxins by binding to them; activate complement proteins in plasma for the destruction of microorganisms by phagocytes; immobilize bacteria; and opsonise various pathogens (Li et al., 2007). IgG, IgM and IgA



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are the major immunoglobulins which are involved in the complement activation, opsonization, neutralization of toxins, etc (Delvis and Roitt, 1998). KA treatment brought in an increase in the number of antibody producing cells as evident from Plaque formation assay and QHS assay heamagglutination titre at doses of 100mg/kg and 200 mg/kg in animal studies. This augmentation of the humoral response to SRBC indicates an enhanced responsiveness of the macrophages and T and B-lymphocyte involved in the antibody synthesis and this might be due to the presence of flavonoids or saponins in the drug which augment the humoral response (Makare et al., 2001). In DTH test, the DTH response, which directly correlates with cell-mediated immunity (CMI), was found to be the highest at the dose 200mg/kg. Increase in the DTH response indicates that Kalpaamruthaa has a stimulatory effect on lymphocytes and necessary cell types required for the expression reaction. The mechanism behind this elevated DTH during the CMI responses could be due to sensitized T-lymphocytes. When challenged by antigen, they undergo proliferation and get converted to lymphoblast` and secrete variety of molecules including pro-inflammatory lymphokines, attracting more scavenger cells to the site of reaction (Delves and Roit, 1998). Sai Ram et al., (2002) have already demonstrated that fruit extract of Emblica relieves the immunosuppressive effects of chromium in rat lymphocytes.

Macrophages play a major role in both nonspecific and specific immune responses. In innate immunity, the phagocytosis of foreign bodies by macrophages render the first barricade against infection and in acquired immunity, they contribute to the regulation of both humoral and cellular immune responses (Kapil and Sharma, 1997). Through interaction with lymphocytes, macrophages play an important role in the initiation and regulation of immune response (Kende, 1982). Most of the plants so far reported for immunomodulatory effects have major affect on nonspecific immunity, i.e. macrophages functions (Dhuley, 1997). Phagocytic index appears to be a close correlate of macrophage activation and of the status of cell mediated response (Saxena et al.,1991). KA caused a significant increase in

phagocytic index of peritoneal macrophages with increase in Kalpaamruthaa doses,indicating its efficiency to upregulate the macrophage activation, thereby proving that Kalpaamruthaa, similar to other different plant based drugs can play an important role in modulating the immune system (Alex et.al,2004)). Besides, the administration of KA significantly ameliorated the total WBCs count, RBCs count and haemoglobin level and also restored the myelosuppressive effects induced by cyclophosphamide in a dose dependent manner. Cyclophosphamide remains the major cancer chemotherapy agent whereas the oxidative stress induced by the drug limits its sustained clinical use (Elangovan et al., 2006). The major drawback of this drug is myelosuppression, which is undesirable,It causes crosslinking of DNA and inhibition of DNA synthesis by acting on both cyclic and intermitotic cells, resulting in general depletion of immune-competent cells. A high degree of cell proliferation renders the bone marrow a sensitive target particularly to cytotoxic drugs. In fact, bone marrow is the organ most affected during any immunosuppression therapy with this class of drugs. Loss of stem cells and inability of the bone marrow to regenerate new blood cells results in thrombocytopenia and leucopenia (Agrawal and Singh, 1999). Administration of KA found to increase the haemoglobin, RBC and WBC count, which was lowered by cyclophosphamide and also it can control the level of myelosupression and thus enhancing the bone marrow activity. Thus, the results of the present study indicates the immunostimulatory activity of Kalpaamruthaa which might be due to the combined action of phytochemicals present in KA on humoral and cell mediated immune responses along with non specific immune response. The efficiency of KA treated animals in overcoming the side effects of drug-induced myelosuppression provides sufficient evidences bringing light on the adaptogenic efficacy of KA. It is not possible at this juncture to single out the most effective immunostimulatory constituent of Kalpaamruthaa. However, based on the published studies, flavonoids seem to be most likely candidates eliciting immunostimulating effect. Thus, Kalpaamruthaa can be a successful candidate for immunotherapy especially in



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combination therapies. Further studies are underway to bring about the molecular mechanism of action.Conflict of InterestNone

ACKNOWLEDGEMENTThe financial support extended by DST

in the form of JRF-PURSE programme to one of the authors Ms. K. Kanchana is gratefully acknowledged.

Table 1: Effect of Kalpaamruthaa on the organ weight of control and experimental animals

Parameters Control KA (50 mg/kg) KA (100 mg/kg) KA (200 mg/Kg)Thymus Weight (mg/100g b.wt.) 0.218 ± 0.01 0.217 ±0.01 0.218 ± 0.01 0.218 ± 0.01Spleen Weight (mg/100g b.wt.) 0.524 ± 0.02 0.529 ± 0.04 0.672 ± 0.05* 0.703 ± 0.04**Liver Weight (g/100g b.wt.) 6.63 ± 0.46 6.85 ± 0.59 7.41 ± 0.56* 7.72 ± 0.54**Kidney Weight (g/100g b.wt.) 1.84 ± 0.13 1.86 ± 0.13 1.89 ± 0.19 1.81 ± 0.13

Data are means ± S. E. of six animals; *p<0.05 and **p<0.01 when compared with the control animals (significantly different).

Table 2: Effect of Kalpaamruthaa on Haematological parameters in control and experimental animals

Parameters Control KA (50mg/kg) KA (100 mg/kg) KA (200 mg/Kg)RBC count (millions/mm3) 7.21 ± 0.43 8.60 ± 0.61 9.30 ± 0.76* 9.87 ± 0.83**

WBC count (thousands/mm3) 5.02 ± 0.39 6.27 ± 0.61 6.55 ± 0.51* 6.73 ± 0.59**Neutrophils (%) 24.65 ±2.71 25.12 ± 2.60 27.73 ± 2.12* 29.44 ± 2.35**

Lymphocytes (%) 61.02 ± 5.03 61.49 ± 4.33 65.84 ± 5.21* 70.13 ± 4.26**


Table 3: Effect of Kalpaamruthaa on the cellularity of lymphoid organs of control and experimental animals

ParametersCellularity (means × 106)

Control KA (50 mg/kg) KA (100 mg/kg) (KA 200 mg/kg)Spleen 388.50 ± 31.06 384.07 ± 30.06 399.63 ± 31.62 458.34 ±35.31**Thymus 77.52 ± 5.78 88.09 ± 6.34 92.9 ± 7.29 126.46 ± 9.19**Bone Marrow 96.08 ± 7.46 98.38 ± 6.99 118.61 ± 8.51* 127.53 ± 8.83**α-Estrase 1178 ± 92.53 1193 ± 96.63 1325 ± 91.80* 1402 ± 97.65**

Data are means ± S.E. of six animals; *p<0.05 and **p<0.01 when compared with the control animals (significantly different).

Table 4: Effect of Kalpaamruthaa on Haemmaglutination titre in control and experimental animals

Groups HA titreGroup I- Control 4.83 ± 0.34

Group II- KA (50 mg/kg) 4.91 ± 0.41Group III- KA (100 mg/kg) 5.18 ± 0.43*Group IV- KA (200 mg/kg) 5.32 ± 0.40**




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Table 5: Effect of Kalpaamruthaa on delayed type of hypersensitivity (DTH) response control and experimental animals

Groups DTH responseGroup I- Control 6.09 ± 0.53Group II- KA (50 mg/kg) 6.43 ± 0.46Group III- KA (100 mg/kg) 7.64 ± 0.59*Group IV- KA (200 mg/kg) 10.51 ± 0.83**

Data are means ± S. E. of six animals; *p<0.05 and **p<0.01 when compared with the control animals (significantly different)

Table 6: Effect of Kalpaamruthaa on cyclosphosphamide induced myelosuppression

Parameters ControlCyclophosphamide (CY)

control (30mg/Kg)CY + KA (50

mg/kg)CY + KA

(100 mg/kg)CY + KA

(200 mg/Kg)RBC

(millions/mm3)10.62±1.01 8.89±0.81a 9.76±0.82 10.19±1.08b 10.52±1.04b

WBC (thousands/mm3)

12.75±1.32 6.68±0.73a 7.13±0.68 9.88±0.98b 11.66±1.59b

Hb (g/dl) 14.09±1.24 9.92±0.87a 11.34±0.98 13.43±1.31b 13.84±1.29b

Data are means ± S. E. of six animals;a *p<0.001 as compared to control

b *p<0.05 as compared to cyclophosphamide control

Figure 1: Effect of Kalpaamruthaa on the humoral immunity as assessed by plaque-forming cell assay




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Figure 2: Effect of Kalpaamruthaa on the humoral immunity as assessed by quantitative hemolysis of SRBC assay


Figure 3: Effect of Kalpaamruthaa on the nonspecific immunity as assessed by the phagocytic capacity


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