Indian Journal of Ayurveda & Research

Immuno-modulatory activity of Rasayana Churna (A Polyherbal Ayurvedic formulation) in  wistar albino  rats
Chaudhari Swapnil1 ,Verma Priyanka2,Galib R3 ,Nariya M4, Patgiri BJ 4,Pajapati PK3
1.Central Ayurveda Research Institute fpr Drug Development ,Kolkata 700091
2.Om Ayurveda and Panchakarma Hospital,Bhavnagar,Gujurat.
3.Dept of Rasashastra and Bhaishajya Kalpana ,All India Institute of Ayurveda,New Delhi,India 110076
4.IPGT &RA ,Gujarat Ayurved University ,Jamnagar,Gujarat 361008

Address for  Coresspondence:
Dr R Galib ,Dept of Rasashastra and Bhaishajya Kalpana,All India Institute of Ayurveda,New Delhi,India-110076.
Email:galib14.yahoo.co.in

ABSTRACT:

Background:Rasayana Churna is a poly-herbal formulation comprising fine powders of Guduchi (Tinospora cordifolia Linn.), Gokshura (Tribulus terrestris Linn.) and Amalaki (Emblica officinalis Linn.) in equal quantities. In Ayurveda, it is expected that the therapeutic efficacy of drug will be increased by levigating with its own juice or decoction. Kupeelu (Strychnos nux-vomica Linn.) is incorporated in this formulation since it is having analgesic, anti-inflammatory and adaptogenic properties. In current work, the immuno-modulatory activity of Rasayana Churna and Bhavita Rasayana Churna each mixed with Kupeelu was evaluated for humoral antibody formation and cell-mediated immunity in suitable experimental models.

Methods:Test drugs were administered orally to the albino rats at a dose of 540mg/kg body weight. Haemagglutination titre, body weight, hematological and histological observations were recorded to assess effects on humoral immunity. Cell-mediated immunity was assessed by immunological paw edema volume.

Results:Both the test drugs have minimal effects on SRBC induced humoral immune response. They did not modify the antibody titre values, hematological values and histo-pathological parameters. Both the drugs produced marked effect on cell mediated immunity response against triple antigen.

Conclusion: Rasayana Churna mixed with Kupeelu have more pronounced effect as compared to Bhavita Rasayana Churna mixed with Kupeelu in suppressing immunological edema.

 KEY WORDS: Haemagglutination titre, Immune-modulatory activity, Rasayana churna, Strychnos nux-vomica

How to cite this article: Chaudari S,Verma P,Galib R,Nariya M ,Patgiri BJ,Prajapati PK .Immuno-modulatory activity of Rasayana Churna (An Ayurvedic compound formulation) in wister albino rats .Indian J Ayurveda Res 2018;1:55-61

Financial Support and Sponsership :Nil

Conflicts of Interest:None

INTRODUCTION:
Ayurveda, one among the traditional knowledge systems is gaining popularity and interest throughout the globe due to its approach in preventive and therapeutic measures.  Alteration in the immune system is caused by various pathological manifestations like rheumatoid arthritis, asthma, allergy, cancer and infectious diseases and the body becomes abnormally susceptible to infections. Immuno-modulation alters the immune system of the host resulting in either immune-stimulation or suppression thus regulating or normalizing it. Hence, immune-modulators are biological response modifiers which improves the host defense mechanism against diseases by striking a balance between regulatory and effector cells.[1,2] Immuno-modulatory agents from plants and animals augment the immune responsiveness of the body against pathogens.[3] The active components of various medicinal plants that regulate the immune system are referred under the heading of Rasayana (immune-modulatory) drugs. They have capability to develop health and longevity, intelligence, memory and youthfulness and improve complexion due to their innate qualities.[4] Ayurveda prescribes many such single drugs and compound formulations, which can be used as potential immunomodulatory drugs. Earlier works on Amalaki Rasayana, Shirishavaleha etc. have proven their immune-modulatory effects.[5,6] Rasayana Churna is a  poly-herbal formulation comprises of Guduchi (Tinospora cordifolia Linn.), Gokshura (Tribulus terrestis Linn.) and Amalaki (Emblica officinalis Linn.) in equal quantities.[7] Its constituents individually are reported to be potent immune-modulators.[8,9,10] This formulation is reported to possess adaptogenic and anti-ulcer activity in experimental models.[11] To incorporate anti-inflammatory and analgesic properties; this formulation has been added with Kupeelu (Strychnos nux-vomica Linn.) having analgesic, anti-inflammatory, apoptotic and anti-tumour activites.[12,13,14] Concept of Bhavana (levigation) is mentioned elaborately in Ayurvedic classics. It is expected that therapeutic efficacy of drug will be increased by levigating with its own juice or decoction. Further this formulation is levigated with decoction prepared from Rasayana Churna to potentiate and prepare Bhavita Rasayana Churna. A comparative immune-modulatory activity of these two formulations was evaluated in current work.

 MATERIALS AND METHODS:

Test formulations
Raw ingredients required for the preparation of test drugs were procured from the pharmacy and were authenticated at Pharmacognosy lab attached with the Institute.  Two test drugs (RCK and BRCK) were prepared by following classical guidelines.
Fine powders (# 72) of individual components of Rasayana Churna (RC) and Kupeelu were blended together, coded as RCK. In the other batch, RC was subjected to three Bhavanas (levigation) with freshly prepared decoction of RC ingredients. After complete drying, it was powdered and powder of Kupeelu was added in it. They are blended together and coded as BRCK.

Experimental Animals
The study was carried out on Charle’s Foster albino rats of either sex, weighing between 200±20g. The animals were procured from the Animal house attached to the Pharmacology laboratory, IPGT&RA, Jamnagar. The animals were kept under ideal husbandry conditions at temperature (22±3°C) with relative humidity (50-70%) and exposed to 12h light and dark cycles each. All animals were exposed to the same environmental conditions and were maintained on standard feed and drinking water ad libitum. The experimental protocol was designed with due permission of Institutional Animal Ethics Committee (IAEC/2012/12/11) and Committee for the Purpose of Control and Supervision on Experiments on Animal (CPCSEA), India guidelines were followed.

Dose fixation
As per Ayurvedic texts, therapeutic dose of Rasayana Churna is 6g per day. Doses of the test drugs (RCK and BRCK) were extrapolated (human dose to animals dose) based on the body surface area ratio referring to the standard table of Paget and Barnes (1969).[15] Both the test drugs were administered orally along with honey and ghee as an adjuvant according to the body weight of the animals with the help of gastric catheter of suitable size sleeved to a syringe nozzle. (Table-1)

 Effect on humoral antibody formation
The effect of the test drugs on antibody formation against SRBC (sheep red blood cells) was measured.[16] The selected animals were categorized into four groups of six animals in each group. The first group (NC) served as the control group and received distilled water. The second group received combination of Madhu and Ghrita (540 mg/kg each) once a day and served as Vehicle control group (VC). Calculated doses of RCK and BRCK with vehicle were administered for 10 consecutive days to the third and fourth groups, respectively. On the third day, sheep blood was collected from the slaughter house in a sterilized bottle containing Alsever’s solution (2% dextrose, 0.8% sodium citrate, 0.5% citric acid, and 0.42% sodium chloride) aseptically to avoid agglutination of blood. The collected sheep blood was thoroughly washed with sterile normal saline through repeated centrifugation until the supernatant fluid became colorless, and was made to 30% SRBC solution. This sensitizing agent was injected subcutaneously at the dose of 0.5 ml/100 g of body weight to the rats of second to fourth groups. On the 11th day, the animals were weighed again and under ether anaesthesia, blood was drawn by puncturing supra-orbital plexus and collected in plain tubes. Serum was separated and complements were inactivated by incubating them for 30 minutes at 56°C temperature in a serological water bath.

Estimation of Haemagglutination antibody titre
The microtitre plate was filled with 0.1 ml sterile normal saline. Complement in the serum was inactivated by heating it at 56°C for 30 minutes in a serological water bath. Serial two-fold dilutions of the serum were performed in sterile saline solution and 0.1 ml of each dilution was aliquoted into 96 well micro titre plates (16 well dilutions for each sample). Blood from the same Sheep was utilized for all sensitization and to determine antibody titre. The covered trays were kept in a refrigerator for overnight and antibody titer was noted on the next day. The titer was converted to log2 values for easy group comparison.[17] [Fig 1]

Fig 1: Antibody titre a) Photograph of microtitre plate, b) Photograph of microtitre plate

 Effect on cell-mediated immune response
Effect on cell-mediated immunity was evaluated using the established procedure.[18] The rats were categorized into four groups of six animals in each group. First group received distilled water and served as normal control group. To the second, third and fourth groups, calculated doses of vehicle, RCK and BRCK were administered for 5 days. All the animals were sensitized subcutaneously (0.5 ml/100 g body weight) on the first day of drug administration using the following solution: triple antigen (DPT) – 1 ml, normal saline (0.9%) – 4 ml, and potash alum (10%) – 1 ml. pH was maintained between 5.6 and 6.8 using 10% sodium carbonate. The drug was administered for five consecutive days. On the fifth day, one hour after drug administration, the initial volume of the left hind paw was noted, and 0.1 ml of the above solution was injected into plantar aponeurosis of same paw. The volume of immunological edema thus produced was measured by the volume displacement method at 24 and 48 hours after the injection using a plethysmograph.[19] Percentage increase in paw volume, the index of edema formation was calculated to assess the cell-mediated immunity response of the drugs.

Statistical analysis
The data was analyzed in terms of mean ± standard error of mean for six rats per experimental group by using unpaired Student’s ‘t’ test to determine statistically significant difference between groups at P < 0.05.

 RESULTS:
Insignificant body weight gain was observed in NC, VC and BRCK groups while the weight was reduced to insignificant extent in RCK group [Table 2]. VC and BRCK significantly increased the spleen weight as compared to NC group but increased weight observed in thymus was statistically insignificant. However, test drugs did not alter spleen, thymus weight and hematological parameters significantly as compared to vehicle group [Table 3, 4]. Microscopic examination of thymus, spleen and lymph node obtained from all groups exhibited normal cyto-architecture [Fig 2,3,4]. Treatment with VC, RCK and BRCK reduced antibody titre; however, the observed decrease did not reach to significant level [Table 5], [Graph 1]. Significant decrease (P<0.05) in paw volume after 24 and 48h was observed in VC compared to control group. In RCK, significant decrease in paw volume was observed after 24h but this decrease level did not reach to statistical significance after 48h recordings. In BRCK group, a decrease in paw volume was observed for both the 24- and 48-hour recordings but did not reach to statistical significance. The difference in decreased paw edema in both treated groups was less compared to control group after 48 hours and values were insignificant (P>0.05) [Table 6], [Graph 2]

Fig 2:Lymph node

Fig3:Spleen

Fig 4:Thymus

Fig 2: Photomicrographs of sections of lymph node taken at x 400 magnification. (a) Normal cytoarchitecture (Normal Control group) (b) Normal cytoarchitecture (Vehicle Control group) (c) Normal cytoarchitecture (RCK group) (d) Normal cytoarchitecture (BRCK group)

Fig 3: Photomicrographs of sections of spleen taken at x 400 magnification. (a) Normal cytoarchitecture (Normal Control group) (b) Normal cytoarchitecture (Vehicle Control group) (c) Normal cytoarchitecture (RCK group) (d) Normal cytoarchitecture (BRCK group)

Fig 4: Photomicrographs of sections of thymus taken at x 400 magnification. (a) Normal cytoarchitecture (Normal Control group) (b) Normal cytoarchitecture (Vehicle Control group) (c) Normal cytoarchitecture (RCK group) (d) Normal cytoarchitecture (BRCK group)

 TABLES:

Table 1: Test drug posology

Group No of animals Drug Dose (mg/kg) Duration
I 6   10 days for humoral antibody formation and
5 days for cell mediated response
II 6 VC (Honey and ghee) 540 mg/kg
III 6 RCK 540 mg/kg
IV 6 BRCK 540 mg/kg

 Table 2: Effect of test drug on body weight of SRBC sensitized albino rats

Treatment Initial body weight (g) Final body weight

(g)

Change in body weight (g)
NC 183.67 ± 12.11 193.33 ± 10.22 5.899 ± 3.67 ↑
VC 214.00 ± 10.63 230.00 ± 9.21 2.539 ± 0.77 ↑
RCK 220.80 ± 8.09 218.00 ± 9.36 4.351 ± 2.14 ↓
BRCK 207.00 ± 12.79 210.00 ± 20.11 8.395 ± 3.93 ↑

Data: Mean ± SEM ↑ – Increase, ↓- Decrease

 Table 3: Effect of test drug on spleen and thymus weight

Treatment Spleen Thymus
Relative weight (g) % change Relative weight (g) % change
NC 0.155± 0.01 ….. 0.148 ± 0.01 …..
VC 0.197± 0.01* 27.09 0.207 ± 0.02 41.89↑
RCK 0.145± 0.01 8.38 0.145 ± 0.01 2.08 ↓
BRCK 0.213± 0.02* 37.41 0.189 ± 0.03 27.70↑

Data: Mean ± SEM ↑ – Increase, ↓- Decrease * P < 0.05 compared to control group

 Table 4: Effect on hematological parameters

Parameters NC VC RCK BRCK
TWBC (103/ml) 8.14 ± 0.99 12.12 ± 2.01 6.10 ± 1.15 7.88 ± 1.06
Neutrophil (%) 36.00 ± 3.728 35.00 ± 5.882 45.60 ± 8.304 37.20 ± 7.883
Lymphocyte (%) 58.40 ± 4.06 64.80 ± 7.70 52.60 ± 8.89 58.20 ± 7.88
Eosinophil (%) 3.00 ± 0.32 2.80 ± 0.20 2.60 ± 0.25 2.40 ± 0.25
Monocyte (%) 2.60 ± 0.25 2.20 ± 0.20 2.00 ± 0.00 2.20 ± 0.20
Hb (g/dl) 12.37 ± 0.99 14.98 ± 0.31* 15.74±0.82* 16.08±0.83**
PCV (%) 38.00 ± 3.93 48.88± 1.64* 50.08± 3.34* 50.30± 2.42*
TRBC (106 /ml) 6.71± 0.66 8.54± 0.39* 8.37± 0.50 * 8.97± 0.41**
MCV (fl) 57.48 ± 0.49 56.16 ± 0.97 58.78 ± 0.42 57.58 ± 0.69
MCH (pg/red cell) 18.32 ± 0.37 17.52 ± 0.73 18.54 ± 0.45 18.02 ± 0.45
MCHC (g/dl) 31.88 ± 0.54 31.30 ± 0.77 31.54 ± 0.59 31.26 ± 0.56
Platelets (103/ml) 1114.20±78.72 1169.20±58.09 1190.20±110.70 1166.80±79.17

Data: Mean ± SEM, ↑- Increase, ↓- Decrease, *P<0.05, **P<0.02 compared to control group

Table 5: Effect of test drug on serum antibody titer of SRBC sensitized albino rats

Treatment Antibody titer (Log2 values) % Change
NC 5.683 ± 0.80
VC 4.990 ± 0.55 12.19 ↓
RCK 3.604 ± 0.59 36.58 ↓
BRCK 4.574 ± 0.71 19.51 ↓

Data: Mean ± SEM, ↓- Decrease

 Table 6: Effect of test drug on immunological paw edema

Treatment % Increase in paw volume
24 hours % inhibition 48 hours % inhibition
NC 38.48 ± 7.58 28.26 ± 3.54
VC 17.17 ± 4.18* 55.36  ↓ 15.17 ± 2.62* 46.30 ↓
RCK 18.96 ± 2.77* 50.70  ↓ 19.82 ± 3.50 29.85 ↓
BRCK 22.35 ± 4.26 41.93  ↓ 24.94 ± 3.46 11.75 ↓

Data: Mean ± SEM, ↑- Increase, ↓- Decrease, *P<0.05

 Graphs

Graph I: Serum antibody Titre of test drugs

Graph II: Effect of Test drugs on immnological paw edema(%)

DISCUSSION:
By cross–linking to form clusters that are more readily ingested by phagocytic cell.  Humoral immunity involves interaction of B cell with the antigen and their subsequent proliferation and differentiation into antibody secreting plasma cells. Antibody functions as the effectors of the humoral response by binding to antigen and neutralizing it or facilitating its elimination BRCK treated rats showed increase in body weight, thymus and spleen weight but found to be statistically insignificant. RCK did not affect any of the parameters studied for humoral immune response. The test drugs and vehicle significantly increased the level of RBC, haemoglobin and PCV which is indicative of their role in red blood cell formation. However, test drugs along with vehicle also increased the above values but did not produce any significant effect when compared with VC indicating that test drugs along with vehicle produce synergistic effects. Though, values of other parameters were increased but found mostly in normal range. The results reveled that both drugs have minimal effects on Sheep RBC induced humoral immune response. Both the test drugs insignificantly influenced T–cell activity which in turn increase vascular permeability, induce vaso-dilatation, macrophage accumulation and activation; and finally result in the increase in the paw volume which promotes phagocytic activity. This behaviour itself is suggestive of activation of immune system at cellular level with lymphocytes, cytokines, prostaglandulin E etc. and also help in liberation from the neighboring cells and the cumulative effect is termed as Delayed Type Hyper-sensitivity (DTH).[20] During DTH responses, sensitized T-lymphocytes, when challenged by the antigen, are converted to lymphoblasts and secret lymphokines, attracting more scavenger cells to the site of reaction.[21] Data obtained during the study shows that the test drugs did not modify antibody formation. This indicates that it is not likely to suppress the formation of IgE type of antibodies. Effect of RCK and BRCK formulations on cell-mediated immunity showed significant suppression of immunological edema. It may have modulatory effect on the IgE-independent, T-lymphocytes-dependent mechanisms. Thus, the RCK and BRCK were found to be attenuating the T-lymphocyte mediated responses, which in turn may be beneficial in the treatment of some of the diseases of the immunological condition like rheumatoid arthritis.

 CONCLUSION:
Both the test drugs have minimal effects on SRBC induced humoral immune response. They did not modify the antibody titre values, hematological values and histo-pathological changes. Suppressant effect was observed in test drugs administered rats on cell-mediated immune response but difference in between the groups is statistically insignificant. RCK has more pronounced effect as compared to BRCK in suppression of immunological edema. Honey and ghee used as a vehicle may synergize the suppression of paw edema effect of test drugs. However, a more detailed systematic study is required to extrapolate results in human beings.

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