Immuno Modulatory Effects of Hot Aqueous Extract of Ocimum sanctum and Argemone mexicana Leaves in Chicken Model  

Puneet Varshney , Sandeep Kumar Dash , Anjana Goel , Ashok Kumar Bhatia
Department of Microbiology and Immunology, College of Veterinary Science and Animal Husbandry, DUVASU, Mathura - 281001, UP, India
Author    Correspondence author
Medicinal Plant Research, 2013, Vol. 3, No. 8   doi: 10.5376/mpr.2013.03.0008
Received: 15 May, 2013    Accepted: 23 May, 2013    Published: 27 May, 2013
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Varshney et al., 2013, Immuno Modulat Effects of Hot Aqueous Extract of Ocimum sanctum and Argemone mexicana Leaves in Chicken Model, Medicinal Plant Research, Vol.3, No.8 57-62 (doi: 10.5376/mpr.2013.03.0008)


The present study was undertaken to study the immuno modulatrory effects of leaves of Ocimum santum (OS) and Argemone mexiana (AM) plants in chicken model. 250 mg/kg body weight oral dose of OS and AM was found ideal and nontoxic in chickens and experimental chickens were fed this dose. Humoral immune response to S. enterica serovar Typhimurium ‘O’ antigen was measured by quantitating the serum antibody level of immunized chickens by ELISA test. There was a significant rise in antibody titre of OS and AM fed chickens in comparison to control group. The antibody titre in the serum samples of HAE of OS and AM fed chickens were (5333.33±674.62) and (4266.67±674.62) respectively, whereas in control group it was 3733.33±533.33. This study indicated that the extract of both plants enhanced the antibody level and acted as a humoral immuno stimulant. Cell mediated immune response (CMI) was assayed by differential lymphocyte count (DLC) and DNCB sensitized hypersensitivity test. DLC revealed increase in lymphocyte count in OS fed group and decrease in lymphocyte count in AM fed group as compared to control group. DNCB test demonstrated 29.89% increase in skin thickness in OS fed group and 34.02% decrease in skin thickness in AM fed group as compared to control group at 24 hour interval. Present study indicated the T cell suppressive impact of hot aqueous extract of AM and stimulator effect of OS.

Argemone mexicana; Ocimum sanctum; Hot aqueous extract; Humoral; CMI; DNCB

There are several diseases which are the result of immunosuppression of either humoral or cell mediated or both type of responses. In such cases often chemical drugs fail to restore the normal functions. Under such circumstances modulation of immune system by medicinal plants or their products could be a possible alternative therapeutic approach. World health organization (WHO) has started to give emphasis on development and use of herbal products for the benefit of world population in viewing the limitations and ill effects of chemical drugs (Lee, 2004). The ecofriendly and non-hazardous nature of herbal medicines for humans and animals, absence of residual effects, minimum problems of drug resistance and absence of side effects further instill the interest in herbal medications.
Among these plants O. sanctum occupies significant place in the indigenous system of medicine of many Asian, African and South American countries. In India the practitioner of traditional system of medicine have used O. sanctum for curing various ailments. From last two decades, to prove the scientific basis of therapeutic use of O. sanctum in modern medicine, several researchers (Sood et al., 2006; Bhartiya et al., 2006; Goel et al., 2010) have begun the exploring the pharmacological effects of this plant and observed that these extracts had modulation effect on the immune responses by regulating cytokines.
Besides these well-established medicinal herbs, there are some weeds like A. mexicana known for their toxicity may have biological activities beneficial to animal kingdom including birds (Kumar, 2006). A. mexicana could be attributed for various disorders scabies, pruritis, eczema and psoriasis, a disease of impaired or defective cell mediated immunity and used in treatment of cataracts and opthalmia (Oudhia, 2001, Gupta, 2004, Kumar, 2006, Goel et al., 2008).
1 Results
1.1 Determination of nontoxic dose
It is well known that seeds of A. mexicana, contain toxic substance called sanguinarine which on consumption produces dropsy in human and animals. However in our study aqueous extract was prepared from A. mexicana leaves, GC-MS & HPTLC of A. mexicana leaves powder revealed the absence of sanguinarine compound. All the three doses of OS and AM were safe and nontoxic as no untoward clinical sign was observed in any of the birds. All the birds were apparently normal and healthy. No adverse effect on hematological parameters was found (data not shown). For humoral and CMI response study OS/AM dose of 250 mg/kg was used as it was ideal.
1.2 Humoral immune responses against S. enterica serovar Typhimurium “O” antigen
Antibody titre in chickens of experimental and control groups against S. enterica serovar Typhimurium ‘O’ antigen was estimated by indirect ELISA. Before immunization the antibody titer in all the three groups was similar ranging from 933.33±133.33 to 933.33±458.12. 7th day after 1st immunization blood sera collected from O. sanctum and A. mexicana extract showed rise in antibody titer to 2800.00±400 and 2266.67±434.1 respectively. The antibody titer further rose to 5333.33±674.62 and 4266.67±674.62 respectively in O. sanctum and A. mexicana fed groups when measured at 7th day after 2nd immunization. The rise in antibody titer was significant in O. sanctum and A. mexicana chickens as compared to control group. The mean values of antibody titer are given in Table 1 and Figure 1.

Table 1 Humoral immune response in chicken against S. enterica serovar Typhimurium “O” antigen

Figure 1 Line diagram showing Humoral Immune response using S. enterica serovar Typhimurium “O” antigen in fed and unfed groups

Cell mediated immune (CMI) response in chickens using DNCB
For studying the effect of HAE of OS and AM leaves on Cell mediated immune response, in vivo assay was employed to assess the delayed type hypersensitivity (DTH) response in chickens using DNCB as an allergen. Mean values of skin thickness of chickens of experimental (OS and AM fed) and control groups at 24 h, 48 h and 72 h post challenge of DNCB are presented in Table 2 and Figure 2. In relation to control group, skin thickness increased markedly in OS fed chickens which were 29.89% higher at 24 h. In AM fed groups thickness was lower than that of control group showing 34.02% inhibition at 24 h. Skin thickness when measured at 48 h and 72 h, OS fed chickens group remained higher 22.98% and 8.10 % increase in comparison to control. In case of AM fed group there was overall inhibition of skin thickness. HAE of OS showed marked cell mediated immune response but AM showed suppression of cell mediated immune response.

Table 2 Mean values of skin thickness in chickens of control, OS and AM fed groups at different time intervals

Figure 2 Line diagram showing difference in skin thickness (mm) in OS and AM fed groups
2 Discussion
Immunomodulatory responses of HAE of OS and AM leaves were evaluated by determining the humoral and cell mediated immune responses. Many workers (Godhwani et al., 1988 and Mediratta et al., 2002) also studied OS effect on immunocytes and found it immunocyte stimulator. Babu et al., (2001) reported that immunomodulatory action is mostly concerned with cellular involvement of haemopoetic and lymphoid tissues. Humoral immune response was quantitated by measuring antibodies by indirect ELISA using S. enterica serovar Typhimurium ‘O’ antigen and results were compared with control (unfed chickens). It was observed that antibody titre in the serum samples of HAE of OS and AM fed chickens were higher (5333.33±674.62) and (4266.67±674.62) respectively, whereas unfed control group exhibited 3733.33±533.33 antibody titre (Table 1 and Figure 1). This indicates that the HAE of OS and AM enhanced the antibody level 42.85% and 14.28% respectively in comparison to control group and acted as humoral response stimulant. These results were reproducible and consistent. Earlier Kumar (2006) also observed similar results using S. enterica serovar Typhimurium antigen in rabbits. Sadekar et al (1998) also demonstrated enhance antibody response in OS leaves fed birds having infection of IBD virus. Maims (2004) reported effective role of OS in enhancing the immune system against bacterial and viral infections. Lombal et al (2004) observed the specific and nonspecific immune responses on account of OS leaves extract and found it to stimulate antibody response. Our results are similar to their findings.
To find the effect of HAE of these plants leaves on cell mediated immune response skin hypersensitivity reaction was done using DNCB as an allergen. In Ocimum sanctum treated group there was 94.73% increase in skin thickness whereas in Argemone mexicana treated group 49.23% increase compared to control group which showed 77.6% increase at 24 hrs. The analysis revealed increase (29.82%, 22.98% and 8.10%) in OS fed group while as decrease (-34.02%, -13.79% and -2.70%) in AM fed chickens as compared to control group after 24, 48 and 72 hrs respectively (Table 2 and Figure 2). Overall results obtained in the present study showed that HAE of OS has stimulatory influence and AM has inhibitory effect on CMI in chickens. This study indicated the T cell suppressive impact of aqueous extract of Argemone mexicana. Recently Goel et al (2010) observed a stimulatory effect of ocimum sanctum on humoral and cell mediated immune response. Humoral immune response observed by increased antibody titre against S. enterica serovar Typhimurium O’ antigen and significant increase in skin thickness by DNCB hypersensitivity test. The stimulant effect of Argemone mexicana on humoral immune response and inhibitory effect on CMI is also reported by Goel et al (2008).
Mediratta et al (2002) investigated the effect of OS seed oil on stressed and non-stressed animals and found that OS modulated both humoral and cell mediated immune response and suggested that this may be due to mediation of GABAergic pathway. In our study HAE of OS was found to have stimulatory effect on both arms of immune system while HAE of AM exhibited antibody stimulation response but caused suppression in CMI response. This study indicates that OS can be used against bacterial, viral infection as well as chicken having immuno suppression. While HAE of AM can be used to treat diseases due to unusual elevated cell mediated immune response (Gupta, 2004; Kumar, 2006; Goel et al., 2008). Mukherjee et al (2005) reported that OS leaves possess some biologically active immunostimulatory components. Goel et al (2010) also found the similar results of Ocimum sanctum as, antiviral and immunomodulator plant on rat, besides reported the increased level of interferon gamma cytokine after orally fed of OS to the experimental rat.
This study conclude that at the present time when we are facing with the limitations of modern medical science in responding to the diseases in chickens due to immuno disorders, phytotherapy offered by the Ayurveda, and other traditional systems of medicine appears to be potential part of the solution. The results conclude that Ocimum sanctum and Argemone mexicana modulate immune response and show promising therapeutic value.
3 Material and Methods
3.1 Preparation of Hot Aqueous Extract (HAE)
The HAE of OS/AM was prepared using Soxhlet apparatus as per the protocol described by Goel et al (2008). The suspension was filtered through muslin cloth and then what man No. 1 filter paper and dried in lyophilizer under vacuum. Percentage yield was 14%~16% (w/w) for OS and 16%~19% (w/w) for AM in terms of dried starting material.
3.2 Experimental birds
Standard pathogen free one day old chicks (Av. Wt 30~35 gm) were purchased from Uday hatchery, Mathura and reared at poultry farm, DUVASU, Mathura. All the birds were housed and fed under standard conditions. Precautions were taken to prevent the environmental contamination. In each experi- mental group, individual bird identification was made by using wing tag. Seven day old chicks were used for the experiments. All these experiments were approved by “Institutional Animal Ethics Committee” (IAEC), and research was conducted under their guidelines. 6 birds per experimental group/control group were used.
3.3 Determination of nontoxic Dose (NTD) of hot aqueous extract (HAE) of O. sanctum and A. mexicana leaves
Hot aqueous extracts (HAE) of O. sanctum/A. mexicana leaves were given orally to chickens. There were three groups for OS as well as for AM for doses, 250 mg/kg, 500 mg/kg and 1000 mg/kg body weight, fed orally for 21 days and one control group which was fed placebo (Triple distilled water) in place of HAE for determination of the safe and nontoxic dose. All the birds were observed for apparent heath condition, toxic signs, hematological changes and change in body weight.
3.4 Humoral immune response
3.4.1 Preparation of S. enterica serovarTyphimurium 'O' antigen
S. enterica serovar Typhimurium “O” antigen was prepared as described by Bhatia et al (2003). Smooth colonies of S. enterica serovar Typhimurium grown on brain heart infusion (BHI) agar medium were selected and inoculated in BHI broth. Inoculated broth was incubated for 6~8 hours at 37ºC and centrifuged at 3000 rpm for 20 minutes and supernatant was discarded and washed with the normal saline and boiled at 100ºC for 2.30 h. Heat inactivated S. enterica serovar Typhimurium culture was employed as ‘O’ antigen for determination of humoral immune response (HIR) in chickens.
3.4.2 Immunization of chickens
Three groups viz; control, O. sanctum and A. mexicana HAE fed, each consisting 6 chickens of age of 7 days were made. OS and AM groups of chickens were fed orally with 250 mg/kg body weight of HAE of OS/AM leaves for 21 days respectively. Control group chickens were given orally triple distilled water (placebo) for 21 days. On 22nd day 1st dose and seven days apart 2nd dose of S. enterica serovar Typhimurium ‘O’ antigen was given subcutaneously to chickens of all the groups as shown in Table 3. Serum samples were collected from each chicken of all the groups before immunization, 7th day after first immunization and 7 days after 2nd immunization for determination of antibody titre against Salmonella ‘O’ antigen by indirect ELISA test.

Table 3 Immunization schedule
3.4.3 Humoral immune response by measurement of antibody titer by indirect ELISA:
The Salmonella “O” antigen coated wells of polystyrene micro titer plate (Nunc) was incubated overnight at 4℃. Following three washings with PBS (PBS: pH 7.2, 0.01M) containing 0.05% tween-20 (PBS-T), blocking was done by 1% bovine serum albumin dissolved in PBS, for 1½ hours at 37℃. Serially diluted serum samples (1:100 to 1:10,240) from 1 to 11 well of micro titer plate respectively 12th well was used as negative control. Rabbit anti chicken immunoglobulin–Y(IgY) conjugated with HRP (Horse radish peroxidase, 1:4000) and substrate solution TMB (1:20) were serially added to all the wells of micro plate, the colour development was stopped after 15 minutes by adding of 50 µL of 1M sulphuric acid to each well. The optical density was measured at 450~570 nm and compared with control group. 

3.5 Cell mediated immune response in chickens using DNCB
This study was designed and conducted to assess the effect of HAE of OS and AM leaves on 2, 4-di nitro chloro benzene (DNCB) sensitized chickens by the method as described by Tiwari and Goel (1985). In this experiment DNCB was used as an antigen. Three groups having 6 chickens (7 day old) in each group were made. Group II and III were fed orally with  250 mg/kg body weight of HAE of OS and AM leaves extract respectively for 21 days. Group I served as control group and placebo (TDW) was given orally for 21 days.
On 22nd day post feeding an aliquot of 0.25 mL of DNCB (10 mg/mL) was applied drop by drop on left side of un-feathered area of abdomen to individual bird in each group and dried by blowing. The vehicle (acetone) alone was applied on right side of abdomen. After 7 days a challenge dose of (1 mg/mL) of DNCB was applied on the same test side and vehicle alone on right side of abdomen (Renu et al., 2003). The skin thickness was measured by Varnier caliper before challenge and 24 h, 48 h, 72 h post-challenge. Difference in the thickness was calculated by subtracting the thickness measured before challenge from that of post challenge.
Authors’ contribution
All the authors contributed equally for this study. All the authors read and approved the final version of the manuscript.
Authors are thankful to the University authorities for providing necessary facilities to carry out this research work.
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