Introduction

Mastitis is one of the most prevalent diseases of dairy animals causing heavy economic loss in terms of quality and quantity of milk (Shem et al., 2002). It involves an inflammatory response in the mammary tissue to multifactorial etio-pathological factors (Mukherjee et al., 2004). The success in the control of mastitis is difficult due to the complex multifactorial etiopathology and multitude of microbial involvements (Sordillo et al., 1997). Antibiotics are the only proven method for treatment of mastitis, however antibiotic therapy of established mammary infection are only moderately efficacious and often fails due to lactation drainage, inflamed glandular cells, immuno suppressions and concurrent infections from dairy environment (Sandholm et al., 1990). Antibiotic therapy further depresses the udder immune system by changing the nature of pathogenic microorganism (Van Den Broek, 1989), formation of resistant bacterial strain, lowering down the oxidative markers for intra cellular killing of microorganism (Hoeben et al.,1997) and contamination of milk in the form of antibiotic residue (Ziv, 1980).Scientists throughout the world are currently investigating the role of alternative medicine to combat this devastating disease. Such ameliorative means are bacterial enzymes, antibacterial peptides, bio response modifiers (Mukherjee et al., 2004), corticosteriod, cytokines (Nickerson et al., 1989), homeopathic formulation, ozone, traditional herbal medicine etc. Nowadays emphasis is also being given to the use of different micronutrients and herbal products to enhance the mammary immunity and to ameliorate the oxidative damage of mammary parenchyma from reactive free radicals (Dash, 2001).WHO has recommended to all member countries to actively promote native medicines of their respective country and initiate steps to conserve medicinal plants. The herbal medicines possess certain advantages being non-toxic, efficacious, cultural acceptability, lesser side effects (Kamboj opcit), and act selectively enhancing body resistance. Jiang et al., 1994, reported herbal preparation containing honey suckle flower, Chrysanthemum morifolium, Citrus reticulata in preventing mastitis. Other medicinal plants like Houttuynia cordata (Hu and Du, 1997), Echium sp., Leptospermum scoparium (Molan, 1996) are also studied in bovine mastitis. Kotle et al. (1999) studied the therapeutic efficacy of Withania somnifera (Ashwagandha), Asperagus racemosus (Shatavari), Garcuma amada (Amahaldi), Ocimum sanctum (Tulsi) in bovine mastitis. Tinospora cardifolia, Ocimum sanctum have also been reported to possess high therapeutic efficacy, anti-inflammatory and immunomodulatory properties (Devi, 1999; Singh, 2000 and Mukherjee, 2001). So far few herbal preparations are in use against mastitis i.e. Mastilep (Saxena et al., 1996), Wisprec, Titali-M (Sharma et al., 1998), Golder udder, udder mint (Allen, 1990) etc. but limited success could be achieved when they are used as adjuvant therapy in mastitis and invariably lack scientific validation. Considering the vast potentiality of plants as sources for antimicrobial drugs the present study was undertaken to screen the antibacterial potential of aqueous extracts of some plants of Kashmir, J&K, India.

Material and Methods

Following herbs (aqueous extract) were evaluated for their In-vivo trial on sub-clinical cases of bovine mastitis.

Plant Material

The selected herbs (leaves) were purchased from registered herbal shops from local market Srinagar, J&K.

Extraction of Plant Material

Plant material was washed with distilled water, dried in shade, grinded to fine powder and stored in airtight container at room temperature in the dark until used. The powdered samples were subjected to extraction using distilled water following the method of Nair et al., 2005.

Preparation of Extracts

Different concentrations of the aqueous extracts of herbals based on safety margin (as per in-vitrocell line toxicity study) and In-vitro AST were prepared.

In vitro Toxicity Study on Cell Line

Measurement of cell viability and proliferation forms the basis for numerous In vitro assays of a cell population’s response to external factors. The MTT Cell Proliferation Assay measures the cell proliferation rate and conversely, when metabolic events lead to apoptosis or necrosis, there is reduction in cell viability. DMEM (Dulbecco’s modified Eagles medium), MTT [3-(4, 5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide], trypsin, EDTA Phosphate Buffered Saline (PBS) and were purchased from Sigma Chemicals Co. (St. Louis, MO) and Fetal Bovine Serum (FBS) were purchased from Gibco. 25 cm ² and 75 cm² flask and 96 well plated purchased from eppendorf India.

Diagnosis of Subclinical Mastitis

The diagnosis of subclinical mastitis (SCM) was based on cow side field tests i.e. California Mastitis Test (CMT) and Somatic Cell Count (SCC) and bacterial isolation. (Table 2)

Collection of Milk Samples

The milk samples from affected quarters from each cow were collected after proper disinfection of teat surface with 70% ethyl alcohol. After teat preparation, 10 ml of milk samples were collected in sterile polyethylene screw capped tubes and kept in box and carried to the laboratory and immediately processed for SCC.

Therapeutic Study

The therapeutic study of following herbs in cases of mastitis that performed well In vitro AST (Table1).The dose of herb was calculated on the following observations-

1) when administered intra-mammary, the herb’s absorption rate is low and less to nil systemic effect is involved and the total dose used is 750 mg as aqueous herbal pure extract,

2) for safety reasons, the maximum dose of a chemical should not be more than 1/10 of the 50% lethal dose (LD50) dose of that particular chemical;

3) the LD50 dose of Fumaria indica aqueous extracts (Gireesh and Vikas Kumar, 2010) was found to be 5 g/kg body weight in rats. Similarly Adiantum capillus LD50 is 500mg/kg b.wt. for 14 days was found to be safe (Gaikwad et al., 2013). Similarly Nepata cataria at doses of 1000–2150 mg/kg BW, did not cause mortality and did not induce any signs of toxicity in the treated male and female mice following dosing (during the observation period of 14 days thereafter), except for the death of one male mouse (Zhu et al., 2009). Thus, to be on the safe side, the doses for all herbal aqueous extracts were taken as 750 mg total used.

Table 1: Therapeutic Study

* are the selected herbs which provide best results on the basis of cell line toxicity and in vitro AST

Statistical Analysis

The data was analyzed using statistical tools (SPSS version 20). ANOVA followed by Duncan’s Multiple Range Test was used for multiple comparisons. Paired‘t’ test was used for pre and post treatment comparisons within each treatment group. Repeated measures ANOVA was used for pre and post treatment multiple comparisons. Statistical differences were determined at the 5% level of significance.

Results and Discussion

Cows treated by aqueous extract of Fumaria indica showed clinical recovery in terms of California Mastitis Test (Table 3) score on day 5 (0.83±0.19) and SCC (Table 4) as compared with day 0 of therapy followed by Adiantum capillus (1.12±0.21) and aqueous extract Nepata cataria (1.33±0.19) of therapy.

Table 2: Correlation between the California Mastitis Test (CMT) and the Somatic Cell Count (SCC)

Table 3: CMT point score in response to cefuroxime and herbal aqueous extracts

Values with different superscript in rows differ significantly (P< 0.05)

The highest antimicrobial activity of the aqueous extract of Fumaria indica could be explained by disturbance of permeability barrier of bacterial membrane structures (Cowan et al., 1999 and Cowan et al., 2008).The moderate antimicrobial activity of aqueous extract of Nepata cataria may be related to the monoterpenoid component i.e. nepetalactone present in the extracts confirmed by a study (Bandh et al. (2011). The CMT point score in group III cows i.e. treated by aqueous extract of Adiantum capillus ranged between 3.47±0.33 to 1.12±0.21. The CMT point score on day 0 was 3.47±0.33 which was significantly (P<0.05) reduced on day 3 to 2.39±0.13 after therapy. The CMT point score significantly reduced to 1.12±0.21 on day 5 by the therapy. The anti-microbial activity of Adiantum capillus can be attributed to the flavonoid constituent of the extract (Brahmachari, 2003). Cows in Group-IV treated by aqueous extract of Nepata catariashowed significant (P<0.05) improvement in California Mastitis Test on day 3 (2.5±0.02) and day 5 (1.33±0.19) as compared with day 0 of therapy which can be attributed to the antimicrobial role of the herbal extracts. The phytochemical screening of the extracts revealed presence of flavonoids, coumarins and glycosides. The presence of flavonoids and a certain degree of lipophilicity might be responsible for the toxicity of the plant leaf extract to the tested microorganisms (Tomas Berberan et al., 1990). This could be as a result of interactions with the membrane constituents and their arrangements. Other authors showed the presence of terpenes and isomers of nepalactones (Bourrel et al., 1993; Recio et al., 1989; Vanden et al., 1991). The CMT point score in group V cows i.e. treated by standard antibiotic ranged between 3.4±0.21 to 0.0±0.00. The CMT point score on day 0 was 3.40±0.21 which was significantly (P<0.05) reduced on day 3 to 1.6±0.32. The CMT point score significantly reduced to 0.0±0.00 on day 5 by the therapy. The SCC point score in group II cows i.e. treated by aqueous extract of Fumaria indica ranged between 54.4±0.15 to 8.3±0.09. The CMT point score day 0 was 54.4±0.15 which was significantly (P<0.05) reduced on day 3 to 40.0±0.94. The CMT point score significantly reduced to 8.3±0.09 on day 5 by the therapy. The SCC point score in group III cows i.e. treated by aqueous extract of Adiantum capillus ranged between 81.13±2.01 to 18.83±1.33. The SCC point score on day 0 was 81.13±2.01 which was significantly (P<0.05) reduced on day 3 to 39.19±2.11. The SCC point score significantly reduced to 18.83±1.33 on day 5 by the therapy. The SCC point score in group IV cows i.e. treated by aqueous extract of Napeta cataria ranged between 78.0±2.1 to 33.33±0.17. The CMT point score on day 0 was 78.0±2.1 which was significantly (P<0.05) reduced on day 3 to 41.0±0.34. The CMT point score significantly reduced to 33.33±0.17 on day 5 by the therapy which can be attributed to the antibacterial role of the aqueous extracts of herbs. Based on CMT the highest clinical recovery was observed in Fumaria extract treated group followed by Adiantum capillus and Nepata cataria.

Table 4: Somatic Cell Count (10⁵ cells/ml of milk) in response cefuroxime and herbal aqueous extract of leaves

Values with different superscript in rows differ significantly (P< 0.05)

Conclusion

In subclinical mastitis significant improvement was observed by using intra-mammary infusion of aqueous extract of Fumaria indica followed by Adiantum capillus and less recovery was noticed by using Nepata cataria. Thus Fumaria indica and Adiantum capillus can be considered as a commercial herbal preparations as an alternative treatment for mastitis.

Acknowledgment

We are thankful to SKUAST-K for providing the necessary facilities to conduct the research. The first author is thankful to the Department of Science and Technology for providing the Inspire fellowship to the author for smooth conduct of research.

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