In vitro efficacy and safety of poly-herbal formulations

Toxicology in Vitro 24 (2010) 885–897

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In vitro efficacy and safety of poly-herbal formulations C.V. Chandrasekaran *, K. Sundarajan, Kripalini David, Amit Agarwal Department of Cellular Assay, R&D Centre, Natural Remedies Pvt. Ltd., Bangalore 560 100, Karnataka, India

a r t i c l e

i n f o

Article history: Received 20 August 2009 Accepted 26 November 2009 Available online 1 December 2009 Keywords: Poly-herbal formulations Cellular antioxidant activity Hepatoprotection Immunomodulation HepG2 cell Ames II™ assay

a b s t r a c t Indigenous plants are used as a traditional source of raw materials for the manufacture of medicines. Modernizing the ancient art of herbal medicine bequeathed from generations entails addressing two interrelated issues i.e. efficacy, and safety prior to their acceptance and use worldwide. The present study was designed to investigate three of our veterinary poly-herbal formulations – Phytocee™ an antistressor; ZigbirÒ a hepatoprotectant; and ZistÒ as an immunomodulator in the pertinent in vitro cell assay models in order to validate their therapeutic potential. Cellular antioxidant potential of Phytocee™ was demonstrated against AAPH induced oxidative stress using HepG2 cells. ZigbirÒ was confirmed as a hepatoprotectant against tert-butylhydroperoxide induced cytotoxicity in HepG2 cells. Immunomodulatory activity of ZistÒ was established by its ability to inhibit the proliferation of mitogen stimulated murine splenocytes in vitro. On treatment with ZistÒ, a trend of decline in IL-6, and IL-12 levels was observed following stimulation with Con A, and LPS respectively in murine splenocytes. Further, all the three poly-herbal formulations were subjected to Ames II™ assay for ensuring their safety profile. Results epitomize that all the three poly-herbal formulations were devoid of significant mutagenic effect in TA98, and TAMix strains of Salmonella typhimurium under our experimental conditions. Ó 2009 Elsevier Ltd. All rights reserved.

1. Introduction In modern period, hub on medicinal plant research has augmented all over the world. Man is turning to natural products; predominantly those derived from plants for his as well as animal’s health care due to the growing recognition that the natural products are non-toxic, lesser side effects, and are accessible at affordable prices. The health status of an animal is mainly influenced by the diet it takes, which in turn affects the performance of the animal. The addition of plant material, especially herbs, in the diets of hens, and broiler chickens can have a positive influence on physiological characteristics viz. egg production, and meat quality (Kroliczewska et al., 2004). In reality, a good nutritional strategy not only perks up the performance of the animal but also guards from various ailments. Ayurveda extensively uses the plant derived compound formulation for the treatment of various ailments. Plants are complex mixtures of compounds and no single compound can provide the desired activity. Some compounds potentiate a desired therapeutic action, while others reinforce the same and yet others interact to

* Corresponding author. Address: Department of Cellular Assay, R&D Centre, Natural Remedies Pvt. Ltd., Plot No. 5B, Veerasandra Industrial Area, 19th K.M. Stone, Hosur Road, Bangalore 560 100, Karnataka, India. Tel.: +91 80 40209999; fax: +91 80 40209817. E-mail addresses: [email protected], [email protected] (C.V. Chandrasekaran). 0887-2333/$ - see front matter Ó 2009 Elsevier Ltd. All rights reserved. doi:10.1016/j.tiv.2009.11.021

neutralize and counteract any possible side effect that may exist. Therefore, several plants with the common desired activities are selected so that the final formulation will have a concentrated desired activity (Jagetia et al., 2004). In this perspective, we at Natural Remedies Pvt. Ltd., Bangalore developed three veterinary poly-herbal formulations i.e. Phytocee™, ZigbirÒ, and ZistÒ encompassing unique combinations of scientifically proven Indian medicinal plants with broad range of pharmacological and clinical benefits for the protection and restoration of health in poultry birds. Phytocee™ is an herbal vitamin C supplement for poultry, a combination of herbs namely Emblica officinalis, Ocimum sanctum, and Withania somnifera. All these plants are reported for their antioxidant property (Fujii et al., 2008; Kath and Gupta, 2006; Bhattacharya et al., 2001). ZigbirÒ is a hepatoprotective poly-herbal poultry feed supplement comprising of Phyllanthus amarus, Andrographis paniculata, Solanum nigrum, and Boerhaavia diffusa. All these plants have widespread traditional use as well as scientific validation as hepatoprotective agents (Naaz et al., 2007; Trivedi and Rawal, 2000; Kuppuswamy et al., 2003; Rawat et al., 1997). ZistÒ, an ideal immunomodulator is a unique blend of herbs like W. somnifera, O. sanctum, E. officinalis, and Mangifera indica. These herbs are known for modulating the immune system (Davis and Kuttan, 2000; Godhwani et al., 1988; Sai Ram et al., 2002; Makare et al., 2001). In the traditional system of Indian medicine, plant formulation and combined extracts of plants are used as drug of choice rather than individual and these herbal formulations are used for the

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treatment of a wide variety of diseases (Ansarullah et al., 2009). This therapeutic approach is often ignored by many and considered to be an alternative to conventional medicine by others due to lack of scientific validation of efficacy and safety (Yuan and Lin, 2000). A recent editorial in JAMA (Journal of American Medical Association) emphasizes that the fundamental issue is not traditional medicine versus alternative medicine, but medical practice supported by clinical and scientific evidence (Fontanarosa and Lundberg, 1998). Hence, there is a requirement for a scientific proof (biological assays, animal models, clinical trials, and chemical standardization). Although it is an ayurvedic poly-herbal preparation, no attempt has been made to scientifically validate its efficacy. More over scientific evidence is available for the individual plants but not for the formulations. Therefore, we attempted to address the efficacy of three poly-herbal preparations (antioxidant activity of Phytocee™ using CAA assay in HepG2 cells; hepatoprotective activity of ZigbirÒ using t-BH induced cytotoxicity in HepG2 cells; immunomodulatory activity of ZistÒ using murine splenocytes) by means of cellular systems. Considering the benefits of these herbal formulations, generating a practical data on their safety is requisite to increase the credibility of their use. Populace equates ‘‘natural” with ‘‘safe” when considering plant-based food supplements. Unfortunately, the assumption that natural products are by definition safe is false. Recent investigations have revealed that some plants used as food or in traditional medicine showed mutagenic effects in, in vitro assays (Higashimoto et al., 1993; Schimmer et al., 1994; Kassie et al., 1996). This raises concern about the potential mutagenic hazards resulting from the use of herbal extracts for animal or human consumption. Subsequently, to ascertain that these formulations are safe for the consumer, their extracts were subjected to in vitro genotoxicity study – Ames II™ assay. 2. Materials and methods 2.1. Chemicals 2-aminoanthracene (2-AA), 4-nitroquinolene-N-oxide (4-NQO), 2-nitrofluorene (2-NF), glucose-6-phosphate (G6P), nicotinamide adenine dinucleotide phosphate (NADP), 20 70 -dichlorofluoresin acetate, quercetin dehydrate, 2,20 -azobis[2-methyl propionamidine] dihydrochloride (AAPH), tert-butylhydroperoxide (t-BH), histopaque 1077, lipopolysaccharide (LPS), and MTT [1-(4,5-dimethylthiazol-2-yl)-3, 5-diphenylformazan] were purchased from Sigma–Aldrich (St. Louis, MO, USA). The metabolic activation system Aroclor™-1254 induced S9 fraction (Sprague Dawley rats) was procured from Moltox (USA). Foetal bovine serum (FBS) was supplied by Hyclone (Logan, USA). The Ames IITM Automated System for high throughput screening (HTS) Kit was purchased from Xenometrix (Allschwil, Switzerland). Earle’s minimum essential media (EMEM), and RPMI-1640 media were obtained from Gibco Life Technologies (Grand Island, NY). Mouse IL-6 ELISA kit OptEIA™, Mouse IL-12 ELISA kit OptEIA™, and cell strainer (40 lm) was purchased from BD Biosciences (USA). All other chemicals and solvents used in this study were of the highest analytical grade available. 2.2. Test substances 2.2.1. Phytocee™ Phytocee™ (Batch No. 0802004) was formulated by totaling E. officinalis fruits (70% w/w), O. sanctum whole plant (20% w/w), and W. somnifera roots (10% w/w). The above poly-herbal mix was subjected to extraction procedure as described below: Phytocee™ was refluxed with methanol in round bottom flask on water bath for 1.5 h at 65–70 °C. Then it was passed through 100 mesh filter cloth. The filtered extract was concentrated and

dried under vacuum at 55–60 °C to yield extract A. The spent obtained from methanolic extraction was dried at room temperature to remove methanol. The dried material was refluxed for 1.5 h with water thrice and passed through 100 mesh filter cloth. The resulting extract was concentrated and dried under vacuum at 80–85 °C to yield extract B. Finally, both the extracts A and B were blended to obtain Phytocee™ extract. 2.2.2. ZigbirÒ ZigbirÒ (Batch No. 01) was prepared by totaling A. paniculata whole plant (27.7% w/w), P. amarus whole plant (27.7% w/w), S. nigrum whole plant (27.7% w/w), and B. diffusa roots (16.9% w/w). The above preparation of ZigbirÒ was extracted using the same procedure as described for Phytocee™. 2.2.3. ZistÒ ZistÒ (Batch No. 47) was primed by adding the herbs at the ratio of 98% crude drug powder and 2% extract form. Crude drug powder contained O. sanctum (46.13% w/w), E. officinalis (18.44% w/w), W. somnifera (27.67% w/w), and bark of M. indica (5.75% w/w). Extracts consisted, 70% methanolic extract of O. sanctum (0.384% w/w), E. officinalis aqueous extract (0.768% w/w), W. somnifera (0.768% w/w), and bark of M. indica (0.05% w/w). This preparation was extracted using the same procedure as described for Phytocee™. 2.3. Cellular antioxidant activity (CAA) assay HepG2 cells (American type culture collection, # HB-8065TM) were cultured in EMEM supplemented with 10% FBS. Stock solutions of Phytocee™ was prepared in DMSO and diluted in culture medium to reach the desired concentrations. The final concentration of DMSO in culture media did not exceed 1% and this media was used as a control. No interference was observed to the test system upon treatment with DMSO up to 1%. Cytotoxicity determination was done by adding Phytocee™ to HepG2 cells and incubating for 24 h prior to assess the antioxidant activity (Wolfe and Liu, 2007). No significant cytotoxic effect was observed with Phytocee™ at concentrations ranging from 31.25 lg/mL to 500 lg/mL to HepG2 cells (data not shown). Then, CAA assay was performed as per the standard method developed by Wolfe and Liu (2007). In this assay, different concentrations of Phytocee™ was pre-incubated with DCFH-DA (25 lM) for 1 h followed by the addition of AAPH (600 lM) free radicals. Quercetin (2–10 lM) was used as reference standard. The fluorescence intensity was measured for 1 h using Fluostar (BMG Labtech) at 37 °C, Excitation 485 nm, Emission 540 nm with a Cycle time 300 s. After blank subtraction from the fluorescent readings, the area under curve (AUC) of fluorescence versus time was integrated to calculate the CAA value at each concentration. EC50 was calculated from the linear regression of the median effect curve (Wolfe and Liu, 2007). 2.4. Hepatoprotection assay HepG2 cells were used to study the protective activity of ZigbirÒ against t-BH induced cytotoxicity. ZigbirÒ was prepared in DMSO and diluted in culture medium to arrive at the desired concentrations. The final concentration of DMSO in culture media did not exceed 1% and this media was used as control. No interference was observed to the test system upon treatment with DMSO up to 1%. Cytotoxicity determination was done by incubating ZigbirÒ with HepG2 cells for 24 h prior to assess the cyto-protective activity (Wolfe and Liu, 2007). No significant cytotoxic effect was noticed with ZigbirÒ at concentrations ranging from 0.1 to 500 lg/mL to HepG2 cells. Hepatoprotection assay was performed as per the method described by Lee et al. (2005) and Thabrew et al. (1997). In this assay, HepG2 cells were pre-incubated with

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Fig. 1. Cellular antioxidant activity (CAA) assay. (A) Peroxyl radical-induced oxidation of DCFH to DCF in HepG2 cells and the inhibition of oxidation by Phytocee™ over time. (B) Median effect plot for inhibition of peroxyl radical-induced DCFH oxidation by Phytocee™. (C) Peroxyl radical-induced oxidation of DCFH to DCF in HepG2 cells and the inhibition of oxidation by quercetin over time. (D) Median effect plot for inhibition of peroxyl radical-induced DCFH oxidation by quercetin.

different concentrations of ZigbirÒ for 2 h. Later, cells were incubated with t-BH (1 mM) and incubated for the following 1 h. At the end of the incubation MTT assay was performed according to

Jover et al. (1992). Silymarin (10–50 lg/mL) was used as a reference standard. The results were expressed as a percentage of protection, calculated as per the below formula by Kinjo et al. (2003):

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Fig. 1 (continued)

%Protection ¼

Mean sample value  Mean of t-BH control Mean of cell control  Mean of t-BH control  100:

2.5. Lymphocyte proliferation and Interleukins assay

ð1Þ

The percentage of cell viability of each treatment was calculated in relation to untreated cells assuming 100% cell viability.

Spleens from Swiss albino mice (8–10 weeks) were removed aseptically and single splenocyte suspension in RPMI-1640 containing 10% heat inactivated FBS, and 1% antibiotics was obtained by passage through 40 lm cell strainer. Lymphocytes were isolated

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by density-gradient centrifugation. Lymphocyte proliferation, and interleukins assays were performed as per the method described by Zhu et al. (2006) with minor modifications. The test substance, ZistÒ was dissolved in DMSO and the final treatment solution contained 0.2% DMSO. No interference was observed to the test system upon treatment with DMSO up to 0.2%. Lymphocytes were seeded at a cell density of 5  105 cells/well and incubated with ZistÒ for 30 min followed by treatment with and without mitogens (LPS – 0.25 lg/mL; ConA – 0.25 lg/mL) in a 48 well cell culture plate. After incubation at 37 °C in a 5% CO2 atmosphere for 48 h, the culture plates were centrifuged and cell supernatants were collected for the estimation of cytokines (IL-6 and IL-12) using commercially available ELISA kits. Proliferative response of the cells was determined by MTT assay. 2.6. Ames II™ assay Ames II™ assay was performed according to Fluckiger-Isler et al. (2004). All the test samples were dissolved in DMSO. DMSO (4%) was used as the solvent control. Cytotoxicity of the extract of poly-herbal formulations was determined in order to allow the selection of appropriate concentrations to test in the mutagenicity assay. The initial cytotoxicity assay was performed with range of concentrations 10–5000 lg/mL using tester strains TA98 and TAMix. No significant cytotoxicity was observed with any of the poly-herbal formulations up to a concentration of 5000 lg/mL (Table 2). The Ames II™ assay was performed with the above concentrations of test Table 1 Cellular antioxidant activity (CAA) assay. Concentration

0 2 lM 4 lM 6 lM 8 lM 10 lM 31.25 lg/mL 62.5 lg/mL 125 lg/mL 250 lg/mL 500 lg/mL

AUC

CAA unit

Phytocee™

Quercetin

Phytocee™

Quercetin

2025 – – – – – 1958 1814 1546 1145 913.5

2304 2194.5 1558.5 792 496.5 342 – – – – –

– – – – – – 3 10 24 43 55

– 5 32 66 78 85 – – – – –

AUC, area under curve; CAA, cellular antioxidant activity.

substances in the presence and absence of Aroclor™-1254 induced rat liver S9 (Moltox, USA). The final concentration of S9 in the assay was 4.5%. S9 mix was incubated with test substance and tester strains for 90 min. Mutagens, 2-NF (2 lg/mL) + 4-NQO (0.5 lg/mL), and 2-AA (5 lg/mL) were used as positive controls in the absence and presence of S9 fraction respectively. The mutagenic activity of the test substance was assessed by considering the criteria narrated in the earlier report (Fluckiger-Isler et al., 2004). 2.7. Statistical analysis All the experiments were performed in three to four replicates per concentration. Results were presented as the mean ± S.D. Statistical differences between the control and treatments were assessed by analysis of variance (ANOVA) followed by Dunnett’s multiple comparison. Statistical significance was arrived for Ames II™ assay using ‘‘t-test” (unpaired, one sided) (Fluckiger-Isler et al., 2004). P < 0.01–0.05 is considered as significant. 3. Results 3.1. Cellular antioxidant activity (CAA) assay The antioxidant potential of Phytocee™ was evaluated by experimenting its ability to prevent AAPH induced oxidation of non-fluorescent 20 70 -dichlorofluorescin diacetate (DCFH-DA) to fluorescent 20 70 -dichlorofluorescin (DCF) in HepG2 cells. Phytocee™ was tested at sub-cytotoxic concentrations up to a maximum concentration of 500 lg/mL in HepG2 cells. Notable dose dependent inhibition of AAPH induced an increase in fluorescence which was observed upon treatment with Phytocee™, and quercetin (Fig. 1A and C). A maximum free radical quenching of 55% at 500 lg/mL of Phytocee™, and 85% at 10 lM of quercetin was observed. CAA unit and AUC at each concentration were calculated and tabulated (Table 1) for the test samples. The median effect plots were generated using the data presented from Phytocee™, and quercetin dose response curves (Fig. 1B and D). 3.2. Hepatoprotection assay The hepatoprotective activity of ZigbirÒ, and Silymarin was assayed with the aid of t-BH induced cytotoxicity in HepG2 cells. A significant dose dependant protection was offered against t-BH

Table 2 Cytotoxicity of the three poly-herbal formulations. Concentration (lg/mL)

S9

DMSO (4%)

TA98 (Mean ± SD)

TAMix (Mean ± SD)

Phytocee™

ZigbirÒ

ZistÒ

Phytocee™

ZigbirÒ

ZistÒ

 +

0.108 ± 0.006 0.222 ± 0.012

0.101 ± 0.008 0.313 ± 0.016

0.107 ± 0.002 0.249 ± 0.011

0.110 ± 0.009 0.223 ± 0.013

0.105 ± 0.014 0.283 ± 0.026

0.101 ± 0.004 0.246 ± 0.014

10

 +

0.104 ± 0.009 0.219 ± 0.010

0.103 ± 0.004 0.320 ± 0.010

0.103 ± 0.049 0.248 ± 0.019

0.105 ± 0.002 0.226 ± 0.016

0.103 ± 0.004 0.291 ± 0.011

0.104 ± 0.014 0.254 ± 0.012

40

 +

0.111 ± 0.014 0.223 ± 0.01

0.101 ± 0.005 0.322 ± 0.014

0.107 ± 0.005 0.269 ± 0.015

0.109 ± 0.002 0.230 ± 0.009

0.103 ± 0.007 0.295 ± 0.008

0.108 ± 0.001 0.265 ± 0.017

140

 +

0.096 ± 0.021 0.223 ± 0.010

0.105 ± 0.001 0.332 ± 0.014

0.104 ± 0.001 0.252 ± 0.009

0.112 ± 0.010 0.227 ± 0.011

0.105 ± 0.010 0.296 ± 0.010

0.097 ± 0.019 0.262 ± 0.015

450

 +

0.096 ± 0.021 0.226 ± 0.011

0.101 ± 0.004 0.335 ± 0.011

0.112 ± 0.010 0.254 ± 0.010

0.118 ± 0.022 0.230 ± 0.007

0.106 ± 0.006 0.289 ± 0.014

0.103 ± 0.008 0.265 ± 0.023

1510

 +

0.092 ± 0.011 0.220 ± 0.001

0.102 ± 0.004 0.331 ± 0.018

0.107 ± 0.010 0.261 ± 0.009

0.107 ± 0.003 0.226 ± 0.009

0.098 ± 0.007 0.275 ± 0.018

0.109 ± 0.002 0.243 ± 0.009

5000

 +

0.099 ± 0.009 0.210 ± 0.007

0.096 ± 0.004 0.302 ± 0.010

0.094 ± 0.002 0.268 ± 0.008

0.104 ± 0.005 0.223 ± 0.006

0.091 ± 0.003 0.264 ± 0.007

0.097 ± 0.009 0.222 ± 0.009

2-NF (2) + 4-NQO (0.5) 2-AA (5)

 +

0.110 ± 0.001 0.205 ± 0.006

0.095 ± 0.001 0.313 ± 0.014

0.096 ± 0.012 0.235 ± 0.016

0.101 ± 0.002 0.216 ± 0.016

0.097 ± 0.013 0.282 ± 0.005

0.079 ± 0.017 0.232 ± 0.009

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Fig. 2. Hepatoprotection assay. (A) Hepatoprotective effects of ZigbirÒ on tert-butyl hydroperoxide induced cell damage in HepG2 cells. Mean of cell control was originally determined to be 457091.25 ± 40719 (lg/ml), which was than normalized to 100%. *P < 0.05 and **P < 0.001 compared to t-BH (1 mM) control. (B) Hepatoprotective effects of Silymarin on tert-butyl hydroperoxide induced cell damage in HepG2 cells. Mean of cell control was originally determined to be 430333 ± 40010 (lg/ml), which was than normalized to 100%. *P < 0.001 compared to t-BH (1 mM) control.

induced damage in HepG2 cells at concentrations of 250, and 500 lg/mL of ZigbirÒ. At the highest concentration (500 lg/mL) of ZigbirÒ, the cell viability was improved significantly up to 50%. The reference standard, Silymarin was tested at concentrations ranging from 10 to 50 lg/mL. The maximum hepatoprotective effect of 31% was observed at the highest concentration (50 lg/mL) of Silymarin (Fig. 2B).

3.3. Lymphocyte proliferation and Interleukins assay In this study, ZistÒ was assessed to find out the cellular basis for its immune-modulating properties. To comprehend this, the effect of ZistÒ was studied in the presence and absence of mitogens using murine splenocytes. In the absence of mitogenic stimulation, ZistÒ had no effect on cell viability of murine splenocytes (data not

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Fig. 3. Lymphocyte proliferation and Interleukins assay. (A) Effect of ZistÒ on murine splenocyte proliferation activated by LPS at 48 h of treatment. *P < 0.01 compared to DMSO (0.2%) + LPS (0.25 lg/ml) control. (B) Effect of ZistÒ on murine splenocyte proliferation activated by Con A at 48 h of treatment. *P < 0.01 compared to DMSO (0.2%) + Con A (0.25) control. (C) Effect of ZistÒ on IL-6 levels in murine splenocytes stimulated in vitro with Con A. *P < 0.01 compared to DMSO (0.2%) + Con A (0.25 lg/ml) control. (D) Effect of ZistÒ on IL-12 levels in murine splenocytes stimulated in vitro with LPS. *P < 0.01 was compared to DMSO (0.2%) + LPS (0.25 lg/ml) control.

shown). No basal levels of IL-6, and IL-12 were detected (data not shown) in ZistÒ treated cell culture supernatants as well as control media (0.2% DMSO). In the next step, mitogen induced proliferation of lymphocytes was examined in association with ZistÒ. LPS (0.25 lg/mL) was used as B cell mitogen, and Con A (0.25 lg/mL) was used as T cell mitogen. At higher concentrations (50 and 100 lg/mL) of ZistÒ, statistically significant decrease in both LPS, and Con A induced lymphocyte proliferation was observed (Fig. 3A and B). Further, cell culture treated supernatants were analyzed for mitogen induced IL-6, and IL-12 release. A significant

dose dependant inhibition of LPS induced IL-12, and Con A induced IL-6 was observed at the highest concentrations of ZistÒ (Fig. 3C and D). 3.4. Ames II™ assay Ames II™ assay was performed in both the absence and presence of metabolic activation to assess the possible mutagenic activity of poly-herbal formulations viz., Phytocee™, ZigbirÒ, and ZistÒ. The fold inductions in revertant histidine colonies upon

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Fig. 3 (continued)

treatment with test substance over the solvent control were found to be less that 2.0 in both TA98, and TAMix S. typhimurium strains. The results demonstrated that none of our poly-herbal formulations exhibited mutagenicity in both TA98, and TAMix S. typhimurium strains. Nevertheless, on treatment with the positive controls (2-NF, 4-NQO, and 2-AA), a significant amplification of the frequency of histidine revertants were observed on cells in the absence and presence of metabolic activation (Tables 3–8).

4. Discussion Feed is a major component, affecting net return from the poultry business, because 80% of the total expenditure in terms of cash is used up on feed purchase (Asghar et al., 2000; Farooq et al.,

2001). Ensuring more net return and minimizing high expenditure on feed are the main challenges, for which many research strategies have been practiced such as introducing feed supplements, and feed additives (Javed et al., 2009). Presently, the scientists are working to improve feed efficiency by means of herbs (Banyapraphatsara, 2007). It was reported that active plant preparations showed promising results in poultry production (Francois, 2006). These plant preparations formed a part of animal diet as preservatives, flavors, digestive enhancers, and remedies for millennia (Deans and Richie, 1987; Piccaglia et al., 1993). Some herbal extracts were shown to be useful in human and veterinary medicine to prevent or alleviate metabolic disorders and their consequences (Wynn, 2001). Also, Sabra and Mehta (1990) applied herbal plants as growth promoters in broiler diets and observed a pronounced improvement in terms of body weight gain, mortal-

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C.V. Chandrasekaran et al. / Toxicology in Vitro 24 (2010) 885–897 Table 3 Mutagenicity testing of Phytocee™ in Salmonella typhimurium strain TA98. Treatment

Concentration (lg/mL)

Number of revertants colonies S9

Replicates 1

Phytocee™

10 40 140 450 1510 5000

*

Mean ± SD 2

3

 +  +  +  +  +  +

2 1 0 2 2 0 1 2 2 1 1 1

0 1 0 0 1 1 1 1 1 1 2 1

1 1 1 0 0 4 1 0 2 0 1 0

1.00 ± 1.00 1.00 ± 0.00 0.33 ± 0.58 0.67 ± 1.15 1.00 ± 1.00 1.67 ± 2.08 1.00 ± 0.00 1.00 ± 1.00 1.67 ± 0.58 0.67 ± 0.58 1.33 ± 0.58 0.67 ± 0.58

Fold increase over baseline 0.55 0.55 0.18 0.37 0.55 0.92 0.55 0.55 0.92 0.37 0.73 0.37

Solvent control

DMSO (4%)

 +

2 0

0 2

0 0

0.67 ± 1.15 0.67 ± 1.15

– –

Positive control

2-NF (2) + 4-NQO (0.5)



48

48

48

48.00 ± 0.00*

26.37

Positive control

2-AA (5)

+

48

48

48

48.00 ± 0.00*

26.37

Mean ± SD 2

3

Fold increase over baseline

P < 0.01.

Table 4 Mutagenicity testing of Phytocee™ in Salmonella typhimurium strain TAMix. Treatment

Concentration (lg/mL)

Number of revertants colonies S9

Replicates 1

Phytocee™

10 40 140 450 1510 5000

*

 +  +  +  +  +  +

2 1 3 1 0 0 0 0 0 0 1 1

2 2 1 1 0 1 1 2 1 2 1 1

0 1 2 0 1 0 2 1 0 0 2 1

1.33 ± 1.15 1.33 ± 0.58 2.00 ± 1.00 0.67 ± 0.58 0.33 ± 0.58 0.33 ± 0.58 1.00 ± 1.00 1.00 ± 1.00 0.33 ± 0.58 0.67 ± 1.15 1.33 ± 0.58 1.00 ± 0.00

0.70 0.54 1.05 0.27 0.17 0.13 0.52 0.40 0.17 0.27 0.70 0.40

Solvent control

DMSO (4%)

 +

1 2

1 0

2 2

1.33 ± 0.58 1.33 ± 1.15

– –

Positive control

2-NF (2) + 4-NQO (0.5)



48

48

48

48.00 ± 0.00*

25.13

Positive control

2-AA (5)

+

45

47

47

46.33 ± 1.15*

18.61

P < 0.01.

ity rate, and feed conversion. As such, more attention has been focused on the utilization of herbal extracts as additive in animal nutrition with the aim to prevent diseases, improve animal welfare as well as reproductive, and productive performances (Viegi et al., 2003). In pursuit of improving poultry production, the growing popularity of herbal medications, easy availability of raw materials, cost effectiveness, and paucity of reported adverse reaction, prompted us to formulate three poly-herbal formulations - Phytocee™, ZigbirÒ, and ZistÒ taking into account the several factors including stress management, and immunity enhancement. Modern poultry are incessantly exposed to a number of stress factors such as heat, overcrowding, handling, exposure to diseases, mycotoxins, and immune system stimulation by vaccination etc. (Deniz et al., 2006). Elevated ambient temperature negatively influences the performance of broilers like reduces feed intake, live weight gain, and feed efficiency (Donkoh, 1989). Several methods are available to alleviate the environmental stressors on the perfor-

mance of poultry. In this respect, a feasible approach to counteract the negative effects of heat stress among chickens would be the dietary supplementation with vitamin C, because of its anti-stress effect (Njoku, 1986; Zapata and Gernat, 1995). Subsequently, we evaluated Phytocee™, an herbal vitamin C supplement in CAA assay. In CAA, Phytocee™ confirmed that it was a good peroxyl radical scavenger with EC50 value of 354 lg/mL. As mentioned earlier, Phytocee™ comprises of botanicals with free radical scavenging, and antioxidant rejuvenating actions. This implies that the antioxidant potential of Phytocee™ can be attributed to its herbal constituents. A study by Javed et al. (2009) cited the improvement in weight gain, feed efficiency, and reduced mortality in broiler chicks by feeding W. somnifera (20 g/L) extract. Narahari et al. (2004) reported that basil leaves incorporated in laying hens diet improved the egg weight, feed efficiency, and the overall health of hens. Several researchers documented the beneficial effects of vitamin C supplementation on growth rate, egg production, eggshell strength, and thickness in stressed poultry (Thornton,

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Table 5 Mutagenicity testing of ZigbirÒ in Salmonella typhimurium strain TA98. Treatment

Concentration (lg/mL)

Number of revertants colonies S9

Replicates 1

ZigbirÒ

10 40 140 450 1510 5000

*

Mean ± SD 2

3

 +  +  +  +  +  +

0 0 3 2 2 2 3 0 1 4 2 6

2 3 3 3 1 2 0 0 0 3 0 2

2 2 1 3 0 0 4 3 1 1 0 3

1.33 ± 1.15 1.67 ± 1.53 2.33 ± 1.15 2.67 ± 0.58 1.00 ± 1.00 1.33 ± 1.15 2.33 ± 2.08 1.00 ± 1.73 0.67 ± 0.58 2.67 ± 1.53 0.67 ± 1.15 3.67 ± 2.08

Fold increase over baseline 0.33 0.67 0.58 1.07 0.25 0.54 0.58 0.40 0.17 1.07 0.17 1.47

Solvent control

DMSO (4%)

 +

2 2

0 0

4 2

2.00 ± 2.00 1.33 ± 1.15

– –

Positive control Positive control

2-NF (2) + 4-NQO (0.5) 2-AA (5)

 +

48 48

48 48

48 48

48.00 ± 0.00* 48.00 ± 0.00*

12.00 19.28

Mean ± SD 2

3

Fold increase over baseline

P < 0.01.

Table 6 Mutagenicity testing of ZigbirÒ in Salmonella typhimurium strain TAMix. Treatment

Concentration (lg/mL)

Number of revertants colonies S9

Replicates 1

ZigbirÒ

10 40 140 450 1510 5000

*

 +  +  +  +  +  +

1 2 1 2 1 3 2 4 1 2 3 2

3 3 1 4 1 3 2 2 2 2 1 1

1 4 3 4 3 5 2 1 1 1 2 1

1.67 ± 1.15 3.00 ± 1.00 1.67 ± 1.15 3.33 ± 1.15 1.67 ± 1.15 3.67 ± 1.15 2.00 ± 0.00 2.33 ± 1.53 1.33 ± 0.58 1.67 ± 0.58 2.00 ± 1.00 1.33 ± 0.58

Solvent control

DMSO (4%)

 +

3 4

0 3

3 1

2.00 ± 1.73 2.67 ± 1.53

Positive control Positive control

2-NF (2) + 4-NQO (0.5) 2-AA (5)

 +

48 48

48 43

48 46

48.00 ± 0.00* 45.67 ± 2.52*

0.45 0.72 0.45 0.80 0.45 0.88 0.54 0.56 0.36 0.40 0.54 0.32 – – 12.87 10.90

P < 0.01.

1962; McDowell, 1989; Bains, 1996). Thus further suggesting that, Phytocee™ would ideally optimize livability, egg production, shell quality, and overall performance of birds. Hepatotoxicity has been viewed as liver injury associated with impaired liver function caused by exposure to drug or other noninfectious agents (Navarro, 2006). Dietary modifications can largely prevent toxic environmental chemicals-induced liver damage. In hepatoprotection assay, it was noted that t-BH induced strong inhibition on cell growth and pretreatment with ZigbirÒ significantly protected HepG2 cells against t-BH induced cytotoxicity. The hepatoprotective potential of ZigbirÒ against t-BH induced damage is due, at least in part, to its direct effects on liver cells. The protective efficacy of ZigbirÒ is reasonably owed to the presence of its individual herbal constituents. Juice of leaves of P. amarus has been used in cholangio-hepatitis, and hepatitis, in animals and has shown hepatoprotective response (Sodhi, 2003). Some of the botanicals used in this formulation have been a part of various well-known hepatoproctive formulations. A. paniculata is one of the herbs used in LIV-O-G, a veterinary poly-herbal hepatoprotec-

tive formulation (Naidu et al., 2007). Apart from hepatoprotective activity, Tipakorn (2002) found that feeding of A. paniculata to broiler chickens improved feed conversion ratio (FCR), live weight, and decreased mortality rate. A. paniculata along with B. diffusa extract forms a part of YAKRIFITÒ, an herbal hepatostimulant, and general tonic in equines (Kumar et al., 1997). S. nigrum is one of the ingredients of ToxiroakÒ, poly-herbal preparation that exhibited protective effects during mycotoxicosis in broilers (Sakhare et al., 2007). Accordingly, the combination of these plants in the formulation of ZigbirÒ may enhance hepatic functions, egg production, growth, weight gain, FCR, livability, and minimizes thin shell, and egg breakage due to liver related problems. The immune system of birds is highly susceptible to the hostile environment and invading pathogens. Stimulation of the immune system by modified feeding can modulate and improve the cellular and humoral immune functions in chicken. In fact, restoration of the normal immune functions may increase resistance to infectious diseases. In ancient Indian literature, several herbal preparations have been described, which can be given to animals to augment

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C.V. Chandrasekaran et al. / Toxicology in Vitro 24 (2010) 885–897 Table 7 Mutagenicity testing of ZistÒ in Salmonella typhimurium strain TA98. Treatment

Concentration (lg/mL)

Number of revertants colonies S9

Replicates 1

ZistÒ

10 40 140 450 1510 5000

*

Mean ± SD 2

3

 +  +  +  +  +  +

1 0 0 1 1 3 0 1 1 3 2 2

0 1 0 2 1 1 1 2 0 0 0 1

1 1 1 3 2 1 1 1 1 2 1 0

0.67 ± 0.58 0.67 ± 0.58 0.33 ± 0.58 2.00 ± 1.00 1.33 ± 0.58 1.67 ± 1.15 0.67 ± 0.58 1.33 ± 0.58 0.67 ± 0.58 1.67 ± 1.53 1.00 ± 1.00 1.00 ± 1.00

Fold increase over baseline 0.73 0.33 0.37 1.00 1.47 0.83 0.73 0.67 0.73 0.83 1.10 0.50

Negative control

DMSO (4%)

 +

0 0

0 1

1 2

0.33 ± 0.58 1.00 ± 1.00

– –

Positive control Positive control

2-NF (2) + 4-NQO (0.5) 2-AA (5)

 +

48 48

48 48

48 48

48.00 ± 0.00* 48.00 ± 0.00*

52.75 24.00

Mean ± SD

Fold increase over baseline

P < 0.01.

Table 8 Mutagenicity testing of ZistÒ in Salmonella typhimurium strain TAMix. Treatment

Concentration (lg/mL)

Number of revertants colonies

10

 +  +  +  +  +  +

1 2 2 1 1 0 0 1 1 3 2 2

0 0 2 2 1 1 1 1 2 0 1 2

2 0 1 1 1 1 0 1 3 0 0 0

1.00 ± 1.00 0.67 ± 1.15 1.67 ± 0.58 1.33 ± 0.58 1.00 ± 0.00 0.67 ± 0.58 0.33 ± 0.58 1.00 ± 0.00 2.00 ± 1.00 1.00 ± 1.73 1.00 ± 1.00 1.33 ± 1.15

S9

Replicates 1

ZistÒ

40 140 450 1510 5000

*

2

3

Solvent control

DMSO (4%)

 +

1 2

1 2

2 0

1.33 ± 0.58 1.33 ± 1.15

Positive control Positive control

2-NF (2) + 4-NQO (0.5) 2-AA (5)

 +

48 46

48 46

48 44

48.00 ± 0.00* 45.33 ± 1.15*

0.52 0.27 0.87 0.54 0.52 0.27 0.17 0.40 1.05 0.40 0.52 0.54 – – 25.13 18.21

P < 0.01.

the immune response (Bhargava and Singh, 1981; Chauhan, 1999). Owing to the immunomodulatory property of ZistÒ, the possible effects that the formulation may have on cells of immune system is quintessential. Lymphocytes constitute the key component of the immune system. A rise or fall in the concentration of these cells affects the health/immune constitution of the body, as they are known to recognize the foreign antigens and mount an immune response. The lymphocyte proliferation assay evinced that ZistÒ was capable of significantly decreasing the in vitro proliferation of mitogen stimulated splenic lymphocytes and thereby inhibiting several immune responses in which these cells are involved. B and T lymphocytes are generally considered the major effector functions associated with cell mediated immunity. These inhibitory effects of ZistÒ on splenocyte proliferation may probably contribute to the suppressive effect on type IV allergy. Since lymphocyte consists of NK cells, T cells, and B cells, ZistÒ induced changes in the percentage of lymphocyte population indicating that ZistÒ might modulate both innate, and adaptive immune functions. Cytokines play crucial roles in regulating various aspects of

immune responses. Inflammatory cytokines such as IL-6 are multi-potential mediators of cellular immune system, having a wide variety of biologic activities. They can have favorable or unfavorable effects on the host immune response, depending on their local concentration. Among cytokines, IL-12 plays a central role in coordinating innate, and cell mediated adaptive immunity. A significant decrease in the levels of IL-6, and IL-12 in murine splenocytes was an indication of the inhibitory action of ZistÒ on both lymphocyte activation, and cytokine production, suggesting anti-inflammatory effects. Several immunological in vitro, and in vivo studies revealed that the total effect of a combination of herbs with immunostimulating potential could be greater than expected from the sum of effects of the single herbs (Wagner and Jurcic, 1991). Synergistic interactions have been documented between different herbs in a formulation (Williamson, 2001). ZistÒ can be described, as an immunomodulator since it possess activity to modulate physiological processes by virtue of its constituents. The immunological potential of the plants we used in the formulation is well-known. Previous studies

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have been reported to support this speculation as the herbals of ZistÒ viz. O. sanctum, E. officinalis, W. somnifera, and M. indica are an active ingredient of StresroakÒ which is used as an anti-stress, immunomodulator, adaptogen, and performance enhancer in poultry (Oyagbemi et al., 2008). In addition, report by Sadekar et al. (1998) suggests the immunopotentiating effect of O. sanctum dry leaf powder on cell mediated immune response in poultry, naturally infected with IBD virus. Given that the use of herbal products for human or animal consumption necessitates the generation of safety data, we screened our three leading poly-herbal formulates viz, Phytocee™, ZigbirÒ, and ZistÒ in Ames II™ assay to assess their mutagenic prospective. Our results unequivocally corroborate that none of our formulations caused mutagenicity in TA98, and TAMix strains in both the absence and presence of S9 fraction as evidenced by the negative results obtained in Ames II™ assay under our experimental conditions. This implies that the formulations neither produced frame shift mutations in TA98 nor base pair mutations in TAMix strains of S. typhimurium. The use of S9 mix solution as a source of metabolic activation system was decisive in order to uncover the plausible risk of formulations after metabolic activation. Since majority of chemical substances/plant extracts are not directly genotoxic in vitro due to the absence of specific metabolic enzymes that are required to trigger the metabolic activation system, which will transform the prodrugs into their metabolically active forms or secondary metabolites (Fluckiger-Isler et al., 2004). From these results, it would be feasible to suggest that the formulations do not contain one or more directly or indirectly acting genotoxic components. Additionally, the safety of each botanical in these formulations is evident from the long history of their safe traditional use in India. Therefore, it is desirable that their combination put to judicious use in the form of herbal formulations will be safe. Nevertheless, the positive controls 2-NF, 4-NQO, and 2-AA significantly amplified the frequency of histidine revertants confirming their mutagenicity (Cui et al., 1999; Nunoshiba and Demple, 1993; Namiki, 1990; Verschaeve et al., 2004; Boobis et al., 1994; Snyderwine et al., 1997). All the above findings lend credence to the beneficial use and safety of Phytocee™, ZigbirÒ, and ZistÒ for overall health of poultry. Nevertheless, the detailed mechanism of their action and the interactions among the different components responsible for their probable activity needs further investigation. Acknowledgement The authors express their heartfelt gratitude to Sri. R.K. Agarwal, Chairman and Managing Director, M/s Natural Remedies Pvt. Ltd., Bangalore, India for his constant inspiration and assistance in successful completion of this work. Authors like to thank the Phytochemistry team for providing the extracts of poly-herbal formulations used in this study. References Ansarullah, Jadeja, R.N., Thounaojam, M.C., Patel, V., Devkar, R.V., Ramachandran, A.V., 2009. Antihyperlipidemic potential of a polyherbal preparation on triton WR 1339 (Tyloxapol) induced hyperlipidemia: a comparison with lovastatin. International Journal of Green Pharmacy 3, 119–124. Asghar, A., Farooq, M., Mian, M.A., Khurshid, A., 2000. Economics of broiler production of Mardan Division. Journal of Rural Development Administration 32, 56–64. Bains, B.S., 1996. The role of vitamin C in stress management. Missset World Poultry 12, 38. Banyapraphatsara, N., 2007. Utilization of medicinal plants in animal production. In: 11th International Congress, Phytopharmacology, Leiden, The Netherlands. Bhargava, K.P., Singh, N., 1981. Antistress activity of Ocimum sanctum. Indian Journal Medicine Research 73, 443.

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