Santhammal et al, JAPHR 2011, Vol 1 Issue 3 Research Article
8
Content list available at: www.iaphp.in
Journal of Advances in Pharmacy and Healthcare Research
Journal home page: http://www.japhr.iaphp.in
ISSN 2231-6817
Evaluation of Immunomodulatory Activity of Saya Churnam (A Poly Herbal Formulation) on Albino rats.
Y. Santhammal1, S. Arthika1, N. Sharly Elgal1, R. Ganesan2, P. Satyarajeswaran2, S.N. Gaidhani2, G.Pramod Reddy2* and T. Anadan2
1Department of Bio-technology, Bishop Heber College, Tirucharappalli, T.N. India.
2Department of Pharmacology, Siddha Central Research Institute, Chennai, T.N., India.
Key words:
Saya churnam, Immunomodulation, Antibody titer,
Delayed type hyper sensitivity, Ig E.
Corresponding Author:
Dr. G. Pramod Reddy, Department of Pharmacology, Siddha Central Research Institute, Anna hospital Campus, Arumbakkam, Chennai, T.N., India.
gpramod01@gmail.com
Abstract:
The immunomodulatory activity of Saya churnam (a poly herbal formulation) on Albino rats was evaluated by using Cyclophosphamide as an immunosuppressant. The Poly herbal formulation was administered orally at the dosage levels of 200mg/kg/day and 400mg/kg/day body weight of rat. The assessment of immunomodulatory activity on specific and non- specific immunity were studied by Hemagglutination antibody (HA) titer, delayed type hypersensitivity (DTH), Identification of Ig E, hematological , biochemical analysis. Induction of immune suppression in rats was achieved by using Cyclophosphamide (CP) (100mg/kg/day, p.o). Oral administration of Saya Churnam (SC) showed a significant increase in the production of circulating antibody titer in response to sheep red blood cells (SRBCs). A significant (p< 0.001) increase in the both primary and secondary HA titer was observed when compared to control group, where as in Cyclophosphamide treated group Saya Churnam showed significant (p<0.01) increase in HA titer. Saya churnam showed significant (p<0.01) Delayed type hypersensitivity (DTH) reaction by facilitating the foot pad thickness response to SRBCs in sensitized rats. Also biochemical and hematological analysis showed a significant (p<0.001) increase in Serum Glutamate Oxaloacetate Transaminase (SGOT), Serum Glutamate Pyruvate Transaminase (SGPT), Alkaline Phosphatase (ALP) and WBCs. The study demonstrates that Saya churnam triggers both specific and non-specific responses to a greater extent. The study showed significantly increased results in Heme agglutination (HA) titer, DTH response, Ig E test, biochemical, hematological analysis.
INTRODUCTION
Indian traditional systems of medicines like Siddha and Ayurveda which generally involve herbal formulations have suggested the body’s natural resistance to disease (Sagrawat and Khan, 2007). Recent studies with plants have revealed many compounds with potent antioxidant, antineoplastic, antiulcer, anti-inflammatory and immunostimulating potential (Wanger, 1990). Immune activation is an
effective as well as protective approach against emerging infectious diseases, and this immune system known to be involved in etiology and pathophysiological mechanisms of several diseases. It is believed that the immunomodulatory drugs promote positive health and maintain organic resistance against infections by establishing body’s equilibrium and conditioning the body tissues (Dasgupta et
Santhammal et al, JAPHR 2011, Vol 1 Issue 3 Research Article
9
al., 1998). The restorative and rejuvenating power of these herbal remedies might be due to their action on the immune system and thereby responsible for the protection of the organism from extraneous substance and maintaining homeostasis.
Plant and plant products are being used as a source of medicine since long. Non – toxic herbal preparations are used to improve the general health by stimulating body’s immunity. In this poly herbal formulation (Terminalia chebula, Piper longum, Piper cubeb, Alpinia galangal, and Myristica officinalis) each herb have a capacity to induce immune response and each herb have anti-inflammatory, anti-ulcer, and other activities.
The Siddha system of medicines not only provides hat alternative, but also scores over the side effects and cost factors of allopathic medicine. Herbal preparation can be more effective and safer (Vinothapooshan et al., 2011) than conventional medicines. Non-toxic herbal drug preparations that are used to improve general health by stimulating body’s immunity (Kumar et al., 1999).
Herbal medicine has become an integral part of standard health care (Agarwal et al., 2010). This formulation of Saya churnam was obtained from the ancient Siddha text (Agasthiar, 1995). In Siddha normally this Saya churnam is prescribed for Tuberculosis, Jaundice, Anemia, Diabetes mellitus, Arthritis. On the basis of literature there are no reports on Saya Churnam for immunomodulatory system.
The present study was undertaken to investigate and to validate the Immunomodulatory activity of Saya churnam.
MATERIALS AND METHODS
Site of the experiment: The experiment was conducted at the Department of Pharmacology, Siddha Central Research Institute, Chennai, in February, 2011.
Plant material: Fruits and rhizome of the plants were procured from local market and it was authentified by Sasikala Ethirajulu, Assistant Director, Department of Pharmacognosy, Siddha Central Research Institute, Chennai.
Preparation of poly herbal formulation: Fruits of Terminalia chebula, Piper longum, Piper cubeb, Myristica officinalis and Rhizome of Alpinia galangal was collected. It was cleaned without any dust particles or other unwanted materials. Each herb is finely powdered and it was mixed in different ratio. The collected fruits and rhizome are fried little and powdered as mentioned in the ancient text. Then the poly herbal powder was used for the further studies. Now this formulation is used for the evaluation study.
Drug and chemicals: All the drugs and chemicals are analytical grade while the other drugs procured were Cyclophosphamide (Biochem pharmaceutical, Mumbai), ELISA commercial kit from Biotran Diagnostics, U.S.A.
Animals: Healthy Wistar albino rats both male and female (180-230 gm) were used for the study. All the animals were housed which were under standard conditions of temperature (25 ± 2? C), 12h light/ dark cycles and fed with standard pellet food and water. The animals were divided into four groups consisting of six animals each. A group of
Santhammal et al, JAPHR 2011, Vol 1 Issue 3 Research Article
10
six un- treated rats were taken as control (group I). Group II animals received standard drug Cyclophosphamide on day 9th and 16th orally in a dosage range of 100mg/kg/day. The Saya churnam was fed orally for 21 days at a dosage of 200 mg/kg/day (group III), 400) mg/kg/day, (group IV) mg/kg/day for assessment of immunomodulatory activity.
Test compound formulations: The different ratio of all the fruit and rhizome powder was measured and prepared in honey with water in the ratio of 7:3 prior to oral administration of animals. Freshly prepared drug solution was used. The vehicle alone served as control.
EXPERIMENTAL PROCEDURE
Antigenic material: Preparation of sheep RBCs (SRBCs): Sheep blood was collected in sterile Alsever’s solution in 2:1 proportion of Alsever’s solution (Freshly prepared). Blood was kept in the refrigerator and processed, for the preparation of SRBCs batch, by centrifugating at 2000rpm for ten minutes and washing with physiological saline 4-5 times and then suspending into buffered saline for further use (Kirtikar et al., 1961).
Heamagglutinating Antibody (HA) Titer: The rats of all groups are pre-treated with drug for 21 days. And each rat immunized with 0.1X109 SRBC/rat by i.p. route, including control rats. The immunization (Agarwal et al., 1999) was given on day 7th and 14th. The animals were treated with Saya churnam for 21 days. The titer was determined by titrating serum dilutions with SRBCs. The micro titer plates were incubated at room temperature for one hour and examined visually for agglutination has been expressed as HA titer.
Delayed Type Hypersensitivity (DTH) Response: Each group of animals (group I-IV) was immunized with SRBCs on day 7th and 14th. On day 21st Rats are administered 0.1ml of 1% SRBCs in the left hind foot pad by subcutaneous (Fulzele et al., 2003) injection, in the right hind foot pad administered 0.1 ml of 0.9% of normal saline was injected and the increased level of paw volume was measured by plethysmometer (Gayathri et al., 2005) in three different time (0 hour, 1 hour, 3 hour) intervals. The thickness between left and right hind paw volume was measured.
Identification of Ig E: On 21st day serum was obtained from all the group of animals. 25µl of serum was added to all the wells, add 100µl of Ig E biotin reagent then allowed to incubation of an hour. Discard the contents and add 300 µl of wash buffer and discarded that it was carried out four times. Then added 100 µl of Ig E enzyme reagent allowed it for incubation of half an hour. Discard the contents and added 300 µl of wash buffer and discarded it was carried out 4 times after that.1 ml of working substrate solution was added allowed for 15 minutes incubation then added 0.05ml of stop solution read the absorbance at 450 nm.
Liver function and blood parameters test: Activities of Serum Glutamate Oxalate Transaminase (Sharififa et al., 2009) (SGOT), Serum Glutamate Pyruvate Transaminase (SGPT), Alkaline Phosphatase (ALP) and Hematological parameters (RBC, WBC, Hb, PLT) were estimated by using kits (Span Diagnostics, India). For this purpose four groups of animals (one control+three
Santhammal et al, JAPHR 2011, Vol 1 Issue 3 Research Article
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Table 1: Effect of Saya Churnam on primary and secondary antibody titer.
Treatment
Primary antibody titer
Secondary antibody titer
Control
6.88±0.83
7.66 ± 0.98
CP 100mg/kg
5.23±0.63**
4.86 ± 1.04**
SCLD 200mg/kg
5.72 ± 1.04***
6.12 ± 0.89***
SCHD 400mg/kg
7.76 ± 1.32***
8.56 ± 0.98***
The values are expressed as (Mean ± S.D), n=6,**p<0.01 and *** p<0.001
Treatments) as described above were used and treated with drug for 21 days.
Statistical analysis: The statistical analysis was performed by using student t Test followed by one way analysis (ANOVA).
RESULTS
Heamagglutination antibody (HA) Titer: Effect of Saya churnam low dose (SCLD) and Saya churnam high dose (SCHD) on primary and secondary antibody response on HA titer is shown in (Table 1). Primary antibody response on day 14th in saya churnam (200mg, 400mg/kg/p.o) treated group with normal immune status showed significant increase (p<0.01) in HA titer when compared with the control group. A significant decrease in the antibody titer was observed in the Cyclophosphamide–treated group when compared with the control group. In immunosuppressed groups, where the immunity was suppressed by administration of Cyclophosphamide on day nine, Saya churnam (400mg/kg/p.o) administration produced a significant (p<0.01) rise in the antibody titer when compared with the Cyclophosphamide – treated group. Secondary antibody titer on twenty first day in Saya churnam both low dose and high dose treated groups with normal immune status group showed a significant rise (p<0.01) in the antibody titer when compared with the control group. In the immunosuppressed groups where the immunity was suppressed by administration of Cyclophosphamide on day sixteenth Saya churnam both low dose and high dose showed a significant rise (p<0.01) in HA titer when compared with the Cyclophosphamide group.
Delayed type hypersensitivity: Effect of Saya churnam on cell mediated immune response by DTH induced foot pad oedema is shown in (Table 2). In the all groups of rats with normal immune status, of Saya churnam low dose (200mg/kg/p.o) and Saya churnam high dose(400mg/kg/p.o) showed significant (**p<0.01,*P<0.05) potentiated DTH response in terms of increase in the mean difference of paw thickness when compared with control group. The drug treated group of rats showed significant (p<0.01) potentiated DTH response in terms of increase in the mean difference of paw thickness. Heightened delayed type hypersensitivity reaction suggests activation of cellular immune system.
Santhammal et al, JAPHR 2011, Vol 1 Issue 3 Research Article
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Table 2: Effect of Saya churnam treatment on cell mediated immune response by delayed type hypersensitivity induced footpad oedema.
Treatment
SRBC’s (in left hind paw)
Saline (in right hind paw)
0 hrs
1 hrs
3 hrs
0 hrs
1 hrs
3 hrs
Control
0.81±0.14
1.14±0.32
2.04±0.33
0.81± 0.15
0.91±0.19
0.96±0.36
SCLD200mg/kg
0.78±0.20**
1.30±0.42**
2.35±0.45**
0.88±0.16*
0.87±0.15*
0.86±0.23**
SCHD400mg/kg
0.98±0.12**
1.36±0.32**
3.64±0.36**
1.00±0.21*
0.65±0.15*
1.05±0.40**
The values are expressed as (Mean ± S.D), n=6.*P<0.05 and **p<0.01.
Identification of Ig E: The administration of Saya churnam both low dose and high dose (200mg/kg/day and 400mg/kg/day) showed significantly increased (*p<0.05) levels of immunoglobulin level when compared to control in the animal serum sample
Table 3: Effect of Saya churnam on Ig E
Treatment
Ig E
Control
16±4.5
SCLD 200 mg/kg
13.6±2.8*
SCHD 400mg/kg
14.3±4.9*
The values are expressed as (Mean ± S.D), n=6.*p < 0.05.
Liver function and test: There was a significant elevation in SGOT, SGPT, ALP as a result of treatment with Saya churnam on both low dose and high dose in this study (***p<0.001,*p<0.01) (Table 4). The drug treated group compared with control group which showed significantly increased values.
Hematological parameter test: In hematological analysis observed significant differences in hematological parameters and drug treated groups are compared with control and Cyclophosphamide treated group. Result shows increased level of WBC, RBC, HGB, PLT on low dose and high dose treated group when compared with control group.
DISCUSSION
Immunostimulatory agents of plant and animal origin enhance the (Fulzele et al., 2003) immune responsiveness of an organism against a pathogen by activating the immune system. However these
poly herbal formulations should be subjected to systematic studies to substantiate the therapeutic
claims made with regard to their clinical utility (Joshi et al., 2003). Immunomodulation is a procedure which can alter the immune system of an organism by interfering with its functions; if it results in an enhancement of immune reaction it is named as immunostimulative drug which primarily implies stimulation of specific and non- specific system. Immunomodulation through natural substances may be considered an alternative for the prevention (Zhang et al., 2011) and cure of disease. Immuno-suppression implies mainly reduce resistance against infections, stress and may occur on account of environmental or chemotherapeutic factor (Makare et al., 2001). The results obtained in the present study indicate that Saya churnam is a potent immunostimulant, stimulating specific and non-specific immune mechanisms.To evaluate the effect of Saya churnam on humoral response, its influence was tested on sheep erythrocyte specific
Santhammal et al, JAPHR 2011, Vol 1 Issue 3 Research Article
13
HA titer in rats (Benaceraf et al., 1978). Cyclophosphamide showed significant inhibition in
Table 4: Effect of Saya churnam on liver enzymes.
Treatment
SGOT U/L
SGPT U/L
ALP U/L
Control
56.42 ± 11.9
19.18 ± 3.72
88.8 ± 16.1
CP 100mg/kg
111.3 ± 18.42***
31.58 ± 9.11**
120.02 ±14.1***
SCLD 200mg/kg
59.73 ± 11.12***
36.68 ± 10.7 **
150.55 ± 16.88 ***
SCHD 400mg/kg
62.85 ± 10.98 ***
47.58 ± 7.06**
270.9 ± 73.65 ***
The values are expressed as (Mean ± S.D), n=6.**p<0.01 and ***p<0.001.
antibody titer response, while saya churnam counteract the suppression of both primary and secondary humoral (Kulkarni et al., 2007)
responses induced by Cyclophosphamide. It indicates that the Saya churnam has the significant effect on humoral antibody response. In DTH test, the DTH response, which directly correlates with cell mediated immunity (CMI), was found to be the highest at maximum dose of 400mg/kg/day tested in this study. Increase in the DTH response indicates that Saya churnam has stimulatory effect on lymphocytes and accessory cell types (Mitra et al., 1999) required for the expression of the reaction Cell mediated immunity the mechanism behind this elevated DTH during the CMI responses could be due to sensitized T- lymphocytes. When challenged by antigen they are converted to lymphoblast and secrete variety of molecules including pro-inflammatory lymphokines, attracting more scavenger cells to the site of reaction (Fulzele et al., 2003).
Increase in the DTH response indicates that the drug has a stimulatory effect on lymphocytes and accessory cell types required for the expression of the reaction (Mitra et al., 1999). Foot volume was enhanced after Saya churnam treatment suggesting cell (Sen et al., 1992) mediated immune enhancement. Immunoglobulin level is a direct measure to detect the humoral immunity. Serum immunoglobulin refers to a group of serum molecules produced by B- lymphocytes, they are soluble and secreted from of B-cell receptors and are produced to a maximum level to counter the invasion by an antigen, and hence they are also called as (Ismail and Asad, 2009) antibodies.
Identification of Ig E also showed the presence Ig E in the tested animals. The results shown significantly increased level of immunoglobulin in Saya churnam treated group when compared with control and Cyclophosphamide treated group. Estimation of LFT enzymes did not show any toxicity effect which was concomitant with any
Table 5: Effect of Saya churnam on Hematological parameters
Groups
WBC
RBC
HGB
PLT
Control
6.7± 3.5
5.32±1.11
9.0 1 ±1.93
406.9±108.3
CP 100mg/kg
3.81± 4.01*
6.96±2.81*
12.42±5.4 *
169.5±200.1**
SCLD 200mg/kg
5.52±2.34 ***
6.61±2.52***
11.32±4.46***
267.2±105.4***
SCHD 400mg/kg
7.63±2.07 ***
6.69±2.33***
11.56±4.43***
342.1±155.7***
The values are expressed as (Mean ± S.D),n=6.*P<0.05, **p<0.01and ***p<0.001.
Santhammal et al, JAPHR 2011, Vol 1 Issue 3 Research Article
14
significant increase in relative weight of liver. High dosage 400mg/kg/day showed increased level of SGPT, SGOT, and ALP. And the results of the presence study revealed that significant difference in the blood parameters. Findings of the present study showed an overall stimulatory effect of Saya churnam on humoral as well as cell-mediated immunity in rats.
CONCLUSION
On the basis of the results obtained in the current study, it can be concluded that the saya churnam has the potential to stimulate cell-mediated and humoral immunity and it may be a potential therapeutic candidate in several immunosuppressed clinical conditions. However, more exhaustive work needs to be performed to substantiate the claim.
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Agarwal, R., S. Diwanay, P. Pakti and B. Patwardhan, 1999. Studies on immunomodulatory activity of Withania somnifera (Ashwagandha) extracts in experimental immune inflammation. J. Ethnopharmacol., 67: 27-35. 10616957
Agasthiar, G.N., 1995. Agasthiar Gowmathi Nool 400. Thamarai Pathippagam., 94.
Benacerraf, B., 1978. A hypothesis to relate the specificity of T lymphocytes and the activity of I region specific Ir genes in macrophages and B lymphocytes. J of Immunology. 120:1809-1812.
Dasgupta, P.S.C. and A. Gomes, 1998. Immunopotentiating activity of immune-21: A poly herbal product. Indian J. Pharmacol., 30:163-168.
Fulzele, S.V., P.M. Satturwar, S.B. Joshi and A.K. Dorle, 2003. Study of the immunomodulatory activity of Haridradi ghrita in rats. Indian J Pharmacology., 35:51-54.
Sharififar, F., S. Pournourmohammadi and M. Arabnejad, 2009. Immunomodulatory activity of aqueous extract of Achillea wilhelmsii C. Koch in mice. Indian J. Exp. Biol., 47: 668-671.
Fulzele, S.V, P.M. Satturwar, S.B. Joshi and A.K. Dorle, 2003. Study of the immunomodulatory activity of Haridradi ghrita in rats. Indian J Pharmacology., 35:51-54.
Gayathri, V., V.V. Asha and A. Subramoniam, 2005. Preliminary studies on the immuno-modulatory and antioxidant properties of selaginella species. Indian J. Pharmacol., 37:381-385.
Joshi, S.B., P.M. Satturwar, S.V.Fulzele and A.K. Dorle, 2003. Study of the immunomodulatory activity of Haridradi ghrita in rats. Indian J Pharmacology., 35:51-54.
Kirtikar, K.R. and B.D. Basu, 1961. Indian Medicinal Plants. Lalit Mohan Basu Publications, Allahabad.
Kulkarni, A.V, B.D. Siraskar, S.S. Jadhav, S.M. Dhonde, A.S. Kulkarni and S.S. Bingi, 2007. Study of hydroalcoholic extract of <I>Portulaca oleracea</I> L. for immunomodulatory activity in mice. Indian J. Green pharmacy., 1,pp:45-49.
Kumar, P.V. R. Kuttan and G. Kuttan, 1999. Effect of “ Rasayanas” a herbal drug preparation on cell-mediated immune responses in tumor bearing mice. Indian J. Exp. Biol., 37:23-26.
Makare, N., S. Bodhankar and V. Rangari, 2001. Immunomodulatory activity of alcoholic extract of Mangifera indica L. in mice. J thnopharmacology., 78:133-137.
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Mitra, S.K, M. Gupta and D.N.K Sharma, 1999. Immunomodulatory effect of IM-133 Phytother Res., 13:341-343.
Sagrawat, H. and Y. Khan, 2007. Immunomodulatory plants: A phytopharmacological. pharmacognosy Rev., 1,pp: 248-260.
Fulzele, S.V., P.M. Satturwar, S.B. Joshi and A.K. Dorle, 2003. Study of the immunomodulatory activity of Haridradi ghrita in rats. Indian J Pharmacology., 35:51-54.
Sen, P., P.K. Mendiratta and A. Ray, 1992. Effects of Azadirachta indica on some biochemical, immunological and visceral parameters in normal and tressed rats. Indian J Exp Biol., 30: 1170-1175
Agarwal, S.S., S.C. Khadase and G.S. Talele, 2010. Studies on immunomodulatory activity of Capparis zeylanica leaf extracts. Int. J. Pharmaceutical sci. Nanotechnol., 3(1): 887-892
Ismail, S. and M. Asad, 2009.Immunomodulatory activity of Acacia catechu. Indian J Physiol Pharmacol., 53(1):25-33.
Vinothapooshan, G. and K. Kumar, 2011. Immunomodulatory activity of various extracts of Adhatoda vasica Linn. in experimental rats. African j. Pharmacy Pharmacl., 5(3):306-310
Wanger, H., 1990. Search for plant derived natural products with Immunostimulatory activity (recent advances). Pure Appl. Chemistry.,62:7:1217-1222.
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8
Content list available at: www.iaphp.in
Journal of Advances in Pharmacy and Healthcare Research
Journal home page: http://www.japhr.iaphp.in
ISSN 2231-6817
Evaluation of Immunomodulatory Activity of Saya Churnam (A Poly Herbal Formulation) on Albino rats.
Y. Santhammal1, S. Arthika1, N. Sharly Elgal1, R. Ganesan2, P. Satyarajeswaran2, S.N. Gaidhani2, G.Pramod Reddy2* and T. Anadan2
1Department of Bio-technology, Bishop Heber College, Tirucharappalli, T.N. India.
2Department of Pharmacology, Siddha Central Research Institute, Chennai, T.N., India.
Key words:
Saya churnam, Immunomodulation, Antibody titer,
Delayed type hyper sensitivity, Ig E.
Corresponding Author:
Dr. G. Pramod Reddy, Department of Pharmacology, Siddha Central Research Institute, Anna hospital Campus, Arumbakkam, Chennai, T.N., India.
gpramod01@gmail.com
Abstract:
The immunomodulatory activity of Saya churnam (a poly herbal formulation) on Albino rats was evaluated by using Cyclophosphamide as an immunosuppressant. The Poly herbal formulation was administered orally at the dosage levels of 200mg/kg/day and 400mg/kg/day body weight of rat. The assessment of immunomodulatory activity on specific and non- specific immunity were studied by Hemagglutination antibody (HA) titer, delayed type hypersensitivity (DTH), Identification of Ig E, hematological , biochemical analysis. Induction of immune suppression in rats was achieved by using Cyclophosphamide (CP) (100mg/kg/day, p.o). Oral administration of Saya Churnam (SC) showed a significant increase in the production of circulating antibody titer in response to sheep red blood cells (SRBCs). A significant (p< 0.001) increase in the both primary and secondary HA titer was observed when compared to control group, where as in Cyclophosphamide treated group Saya Churnam showed significant (p<0.01) increase in HA titer. Saya churnam showed significant (p<0.01) Delayed type hypersensitivity (DTH) reaction by facilitating the foot pad thickness response to SRBCs in sensitized rats. Also biochemical and hematological analysis showed a significant (p<0.001) increase in Serum Glutamate Oxaloacetate Transaminase (SGOT), Serum Glutamate Pyruvate Transaminase (SGPT), Alkaline Phosphatase (ALP) and WBCs. The study demonstrates that Saya churnam triggers both specific and non-specific responses to a greater extent. The study showed significantly increased results in Heme agglutination (HA) titer, DTH response, Ig E test, biochemical, hematological analysis.
INTRODUCTION
Indian traditional systems of medicines like Siddha and Ayurveda which generally involve herbal formulations have suggested the body’s natural resistance to disease (Sagrawat and Khan, 2007). Recent studies with plants have revealed many compounds with potent antioxidant, antineoplastic, antiulcer, anti-inflammatory and immunostimulating potential (Wanger, 1990). Immune activation is an
effective as well as protective approach against emerging infectious diseases, and this immune system known to be involved in etiology and pathophysiological mechanisms of several diseases. It is believed that the immunomodulatory drugs promote positive health and maintain organic resistance against infections by establishing body’s equilibrium and conditioning the body tissues (Dasgupta et
Santhammal et al, JAPHR 2011, Vol 1 Issue 3 Research Article
9
al., 1998). The restorative and rejuvenating power of these herbal remedies might be due to their action on the immune system and thereby responsible for the protection of the organism from extraneous substance and maintaining homeostasis.
Plant and plant products are being used as a source of medicine since long. Non – toxic herbal preparations are used to improve the general health by stimulating body’s immunity. In this poly herbal formulation (Terminalia chebula, Piper longum, Piper cubeb, Alpinia galangal, and Myristica officinalis) each herb have a capacity to induce immune response and each herb have anti-inflammatory, anti-ulcer, and other activities.
The Siddha system of medicines not only provides hat alternative, but also scores over the side effects and cost factors of allopathic medicine. Herbal preparation can be more effective and safer (Vinothapooshan et al., 2011) than conventional medicines. Non-toxic herbal drug preparations that are used to improve general health by stimulating body’s immunity (Kumar et al., 1999).
Herbal medicine has become an integral part of standard health care (Agarwal et al., 2010). This formulation of Saya churnam was obtained from the ancient Siddha text (Agasthiar, 1995). In Siddha normally this Saya churnam is prescribed for Tuberculosis, Jaundice, Anemia, Diabetes mellitus, Arthritis. On the basis of literature there are no reports on Saya Churnam for immunomodulatory system.
The present study was undertaken to investigate and to validate the Immunomodulatory activity of Saya churnam.
MATERIALS AND METHODS
Site of the experiment: The experiment was conducted at the Department of Pharmacology, Siddha Central Research Institute, Chennai, in February, 2011.
Plant material: Fruits and rhizome of the plants were procured from local market and it was authentified by Sasikala Ethirajulu, Assistant Director, Department of Pharmacognosy, Siddha Central Research Institute, Chennai.
Preparation of poly herbal formulation: Fruits of Terminalia chebula, Piper longum, Piper cubeb, Myristica officinalis and Rhizome of Alpinia galangal was collected. It was cleaned without any dust particles or other unwanted materials. Each herb is finely powdered and it was mixed in different ratio. The collected fruits and rhizome are fried little and powdered as mentioned in the ancient text. Then the poly herbal powder was used for the further studies. Now this formulation is used for the evaluation study.
Drug and chemicals: All the drugs and chemicals are analytical grade while the other drugs procured were Cyclophosphamide (Biochem pharmaceutical, Mumbai), ELISA commercial kit from Biotran Diagnostics, U.S.A.
Animals: Healthy Wistar albino rats both male and female (180-230 gm) were used for the study. All the animals were housed which were under standard conditions of temperature (25 ± 2? C), 12h light/ dark cycles and fed with standard pellet food and water. The animals were divided into four groups consisting of six animals each. A group of
Santhammal et al, JAPHR 2011, Vol 1 Issue 3 Research Article
10
six un- treated rats were taken as control (group I). Group II animals received standard drug Cyclophosphamide on day 9th and 16th orally in a dosage range of 100mg/kg/day. The Saya churnam was fed orally for 21 days at a dosage of 200 mg/kg/day (group III), 400) mg/kg/day, (group IV) mg/kg/day for assessment of immunomodulatory activity.
Test compound formulations: The different ratio of all the fruit and rhizome powder was measured and prepared in honey with water in the ratio of 7:3 prior to oral administration of animals. Freshly prepared drug solution was used. The vehicle alone served as control.
EXPERIMENTAL PROCEDURE
Antigenic material: Preparation of sheep RBCs (SRBCs): Sheep blood was collected in sterile Alsever’s solution in 2:1 proportion of Alsever’s solution (Freshly prepared). Blood was kept in the refrigerator and processed, for the preparation of SRBCs batch, by centrifugating at 2000rpm for ten minutes and washing with physiological saline 4-5 times and then suspending into buffered saline for further use (Kirtikar et al., 1961).
Heamagglutinating Antibody (HA) Titer: The rats of all groups are pre-treated with drug for 21 days. And each rat immunized with 0.1X109 SRBC/rat by i.p. route, including control rats. The immunization (Agarwal et al., 1999) was given on day 7th and 14th. The animals were treated with Saya churnam for 21 days. The titer was determined by titrating serum dilutions with SRBCs. The micro titer plates were incubated at room temperature for one hour and examined visually for agglutination has been expressed as HA titer.
Delayed Type Hypersensitivity (DTH) Response: Each group of animals (group I-IV) was immunized with SRBCs on day 7th and 14th. On day 21st Rats are administered 0.1ml of 1% SRBCs in the left hind foot pad by subcutaneous (Fulzele et al., 2003) injection, in the right hind foot pad administered 0.1 ml of 0.9% of normal saline was injected and the increased level of paw volume was measured by plethysmometer (Gayathri et al., 2005) in three different time (0 hour, 1 hour, 3 hour) intervals. The thickness between left and right hind paw volume was measured.
Identification of Ig E: On 21st day serum was obtained from all the group of animals. 25µl of serum was added to all the wells, add 100µl of Ig E biotin reagent then allowed to incubation of an hour. Discard the contents and add 300 µl of wash buffer and discarded that it was carried out four times. Then added 100 µl of Ig E enzyme reagent allowed it for incubation of half an hour. Discard the contents and added 300 µl of wash buffer and discarded it was carried out 4 times after that.1 ml of working substrate solution was added allowed for 15 minutes incubation then added 0.05ml of stop solution read the absorbance at 450 nm.
Liver function and blood parameters test: Activities of Serum Glutamate Oxalate Transaminase (Sharififa et al., 2009) (SGOT), Serum Glutamate Pyruvate Transaminase (SGPT), Alkaline Phosphatase (ALP) and Hematological parameters (RBC, WBC, Hb, PLT) were estimated by using kits (Span Diagnostics, India). For this purpose four groups of animals (one control+three
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Table 1: Effect of Saya Churnam on primary and secondary antibody titer.
Treatment
Primary antibody titer
Secondary antibody titer
Control
6.88±0.83
7.66 ± 0.98
CP 100mg/kg
5.23±0.63**
4.86 ± 1.04**
SCLD 200mg/kg
5.72 ± 1.04***
6.12 ± 0.89***
SCHD 400mg/kg
7.76 ± 1.32***
8.56 ± 0.98***
The values are expressed as (Mean ± S.D), n=6,**p<0.01 and *** p<0.001
Treatments) as described above were used and treated with drug for 21 days.
Statistical analysis: The statistical analysis was performed by using student t Test followed by one way analysis (ANOVA).
RESULTS
Heamagglutination antibody (HA) Titer: Effect of Saya churnam low dose (SCLD) and Saya churnam high dose (SCHD) on primary and secondary antibody response on HA titer is shown in (Table 1). Primary antibody response on day 14th in saya churnam (200mg, 400mg/kg/p.o) treated group with normal immune status showed significant increase (p<0.01) in HA titer when compared with the control group. A significant decrease in the antibody titer was observed in the Cyclophosphamide–treated group when compared with the control group. In immunosuppressed groups, where the immunity was suppressed by administration of Cyclophosphamide on day nine, Saya churnam (400mg/kg/p.o) administration produced a significant (p<0.01) rise in the antibody titer when compared with the Cyclophosphamide – treated group. Secondary antibody titer on twenty first day in Saya churnam both low dose and high dose treated groups with normal immune status group showed a significant rise (p<0.01) in the antibody titer when compared with the control group. In the immunosuppressed groups where the immunity was suppressed by administration of Cyclophosphamide on day sixteenth Saya churnam both low dose and high dose showed a significant rise (p<0.01) in HA titer when compared with the Cyclophosphamide group.
Delayed type hypersensitivity: Effect of Saya churnam on cell mediated immune response by DTH induced foot pad oedema is shown in (Table 2). In the all groups of rats with normal immune status, of Saya churnam low dose (200mg/kg/p.o) and Saya churnam high dose(400mg/kg/p.o) showed significant (**p<0.01,*P<0.05) potentiated DTH response in terms of increase in the mean difference of paw thickness when compared with control group. The drug treated group of rats showed significant (p<0.01) potentiated DTH response in terms of increase in the mean difference of paw thickness. Heightened delayed type hypersensitivity reaction suggests activation of cellular immune system.
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Table 2: Effect of Saya churnam treatment on cell mediated immune response by delayed type hypersensitivity induced footpad oedema.
Treatment
SRBC’s (in left hind paw)
Saline (in right hind paw)
0 hrs
1 hrs
3 hrs
0 hrs
1 hrs
3 hrs
Control
0.81±0.14
1.14±0.32
2.04±0.33
0.81± 0.15
0.91±0.19
0.96±0.36
SCLD200mg/kg
0.78±0.20**
1.30±0.42**
2.35±0.45**
0.88±0.16*
0.87±0.15*
0.86±0.23**
SCHD400mg/kg
0.98±0.12**
1.36±0.32**
3.64±0.36**
1.00±0.21*
0.65±0.15*
1.05±0.40**
The values are expressed as (Mean ± S.D), n=6.*P<0.05 and **p<0.01.
Identification of Ig E: The administration of Saya churnam both low dose and high dose (200mg/kg/day and 400mg/kg/day) showed significantly increased (*p<0.05) levels of immunoglobulin level when compared to control in the animal serum sample
Table 3: Effect of Saya churnam on Ig E
Treatment
Ig E
Control
16±4.5
SCLD 200 mg/kg
13.6±2.8*
SCHD 400mg/kg
14.3±4.9*
The values are expressed as (Mean ± S.D), n=6.*p < 0.05.
Liver function and test: There was a significant elevation in SGOT, SGPT, ALP as a result of treatment with Saya churnam on both low dose and high dose in this study (***p<0.001,*p<0.01) (Table 4). The drug treated group compared with control group which showed significantly increased values.
Hematological parameter test: In hematological analysis observed significant differences in hematological parameters and drug treated groups are compared with control and Cyclophosphamide treated group. Result shows increased level of WBC, RBC, HGB, PLT on low dose and high dose treated group when compared with control group.
DISCUSSION
Immunostimulatory agents of plant and animal origin enhance the (Fulzele et al., 2003) immune responsiveness of an organism against a pathogen by activating the immune system. However these
poly herbal formulations should be subjected to systematic studies to substantiate the therapeutic
claims made with regard to their clinical utility (Joshi et al., 2003). Immunomodulation is a procedure which can alter the immune system of an organism by interfering with its functions; if it results in an enhancement of immune reaction it is named as immunostimulative drug which primarily implies stimulation of specific and non- specific system. Immunomodulation through natural substances may be considered an alternative for the prevention (Zhang et al., 2011) and cure of disease. Immuno-suppression implies mainly reduce resistance against infections, stress and may occur on account of environmental or chemotherapeutic factor (Makare et al., 2001). The results obtained in the present study indicate that Saya churnam is a potent immunostimulant, stimulating specific and non-specific immune mechanisms.To evaluate the effect of Saya churnam on humoral response, its influence was tested on sheep erythrocyte specific
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HA titer in rats (Benaceraf et al., 1978). Cyclophosphamide showed significant inhibition in
Table 4: Effect of Saya churnam on liver enzymes.
Treatment
SGOT U/L
SGPT U/L
ALP U/L
Control
56.42 ± 11.9
19.18 ± 3.72
88.8 ± 16.1
CP 100mg/kg
111.3 ± 18.42***
31.58 ± 9.11**
120.02 ±14.1***
SCLD 200mg/kg
59.73 ± 11.12***
36.68 ± 10.7 **
150.55 ± 16.88 ***
SCHD 400mg/kg
62.85 ± 10.98 ***
47.58 ± 7.06**
270.9 ± 73.65 ***
The values are expressed as (Mean ± S.D), n=6.**p<0.01 and ***p<0.001.
antibody titer response, while saya churnam counteract the suppression of both primary and secondary humoral (Kulkarni et al., 2007)
responses induced by Cyclophosphamide. It indicates that the Saya churnam has the significant effect on humoral antibody response. In DTH test, the DTH response, which directly correlates with cell mediated immunity (CMI), was found to be the highest at maximum dose of 400mg/kg/day tested in this study. Increase in the DTH response indicates that Saya churnam has stimulatory effect on lymphocytes and accessory cell types (Mitra et al., 1999) required for the expression of the reaction Cell mediated immunity the mechanism behind this elevated DTH during the CMI responses could be due to sensitized T- lymphocytes. When challenged by antigen they are converted to lymphoblast and secrete variety of molecules including pro-inflammatory lymphokines, attracting more scavenger cells to the site of reaction (Fulzele et al., 2003).
Increase in the DTH response indicates that the drug has a stimulatory effect on lymphocytes and accessory cell types required for the expression of the reaction (Mitra et al., 1999). Foot volume was enhanced after Saya churnam treatment suggesting cell (Sen et al., 1992) mediated immune enhancement. Immunoglobulin level is a direct measure to detect the humoral immunity. Serum immunoglobulin refers to a group of serum molecules produced by B- lymphocytes, they are soluble and secreted from of B-cell receptors and are produced to a maximum level to counter the invasion by an antigen, and hence they are also called as (Ismail and Asad, 2009) antibodies.
Identification of Ig E also showed the presence Ig E in the tested animals. The results shown significantly increased level of immunoglobulin in Saya churnam treated group when compared with control and Cyclophosphamide treated group. Estimation of LFT enzymes did not show any toxicity effect which was concomitant with any
Table 5: Effect of Saya churnam on Hematological parameters
Groups
WBC
RBC
HGB
PLT
Control
6.7± 3.5
5.32±1.11
9.0 1 ±1.93
406.9±108.3
CP 100mg/kg
3.81± 4.01*
6.96±2.81*
12.42±5.4 *
169.5±200.1**
SCLD 200mg/kg
5.52±2.34 ***
6.61±2.52***
11.32±4.46***
267.2±105.4***
SCHD 400mg/kg
7.63±2.07 ***
6.69±2.33***
11.56±4.43***
342.1±155.7***
The values are expressed as (Mean ± S.D),n=6.*P<0.05, **p<0.01and ***p<0.001.
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significant increase in relative weight of liver. High dosage 400mg/kg/day showed increased level of SGPT, SGOT, and ALP. And the results of the presence study revealed that significant difference in the blood parameters. Findings of the present study showed an overall stimulatory effect of Saya churnam on humoral as well as cell-mediated immunity in rats.
CONCLUSION
On the basis of the results obtained in the current study, it can be concluded that the saya churnam has the potential to stimulate cell-mediated and humoral immunity and it may be a potential therapeutic candidate in several immunosuppressed clinical conditions. However, more exhaustive work needs to be performed to substantiate the claim.
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