Accepted Manuscript

Bovine mastitis: An appraisal of its alternative herbal cure

Saleem Mushtaq, Aabid Manzoor Shah, Aiyatullah Shah, Sajad Ahmad Lone,
Aehtesham Hussain, Qazi Parvaiz Hassan, Md Niamat Ali

PII: S0882-4010(17)31063-X
DOI: 10.1016/j.micpath.2017.12.024
Reference: YMPAT 2660

To appear in: Microbial Pathogenesis

Received Date: 24 August 2017

Revised Date: 7 December 2017
Accepted Date: 8 December 2017

Please cite this article as: Mushtaq S, Shah AM, Shah A, Lone SA, Hussain A, Hassan QP, Ali MN,
Bovine mastitis: An appraisal of its alternative herbal cure, Microbial Pathogenesis (2018), doi: 10.1016/
j.micpath.2017.12.024.

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1 Bovine mastitis: An appraisal of its alternative herbal cure

2 Saleem Mushtaq1, 2, Aabid Manzoor Shah1, Aiyatullah Shah1, Sajad Ahmad Lone1, Aehtesham
3 Hussain1, Qazi Parvaiz Hassan1, *, Md Niamat Ali2
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4 Biotechnology Division, CSIR – Indian Institute of Integrative Medicine, Sanatnagar, Srinagar,
5 Jammu & Kashmir 190005, India

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6 Centre of Research for Development (CORD), University of Kashmir, Hazratbal, Srinagar,
7 Jammu & Kashmir 190006, India

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9 * Corresponding author details: Dr. Qazi Parvaiz Hassan, Senior Scientist, Biotechnology

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10 Division, CSIR – Indian Institute of Integrative Medicine, Sanatnagar, Srinagar, Jammu &
11 Kashmir 190005, India

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12 Email Id. qphassan@iiim.ac.in, pervaizqazi@yahoo.com
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15 Saleem Mushtaq ORCID ID: orcid.org/0000-0002-4961-9723
16 Aiyatullah Shah ORCIDID: orcid.org/0000-0001-9624-4365
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17 Qazi Parvaiz Hassan ORCID ID: orcid.org/0000-0001-9602-3092

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29 Abstract: Bovine mastitis is globally recognised as the most common and costly disease
30 affecting dairy herds. The disease causes huge financial losses to dairy industries by reduced
31 yield and milk quality, deaths and culling of affected cows and also by associated treatment
32 costs. The disease occurs due to invasion of the mammary glands by pathogenic bacteria
33 followed by their multiplication in the milk producing tissues. The most common treatment

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34 method available against bovine mastitis is the intra-mammary infusion of antibiotics. However,
35 their use is associated with the problem of antimicrobial resistance. This scenario has made

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36 search for alternative treatment approaches necessary. Medicinal plants with their well-
37 established history are an excellent natural product resource used as an alternative therapy.

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38 Antibacterial agents from plants can act as important sources of novel antibiotics, efflux pump

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39 inhibitors, compounds that target bacterial virulence or can be used in combination with existing
40 drugs. The plants form an essential component of ethno-veterinary medicine used in the
41 treatment of different diseases like bovine mastitis. This review article attempts to provide an
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42 overview of the different medicinal plants used in the treatment of bovine mastitis. Antimicrobial
43 studies of these plant species and some of their isolated constituents have been reviewed in
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44 detail. It highlights the logic and precedence behind mining this important natural product
45 resource. Our own research findings in this direction and future scope of research are also
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46 discussed briefly.
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47 Keywords: Bovine mastitis, antibiotic resistance, medicinal plants, dairy, antibacterial

48 1.1. Introduction
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49 Bovine mastitis is the inflammation of the mammary glands in dairy cows, caused by invasion
50 and destruction of the milk producing tissues by pathogenic microorganisms [1]. About 137
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51 species of microorganisms including bacteria, yeasts and algae are known to cause mastitis [2,
52 3]. Mastitis causes financial losses to dairy industries through reduced milk yield, poor milk
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53 quality, increased culling and also due to additional expenses on treatment and extra labor [4].
54 India is the world’s largest milk producer, with 16 percent of global production [5]. The annual
55 economic losses due to mastitis in India, United States, UK and worldwide have been estimated
56 at $1.1 billion [6], $2 billion [7-8], $371 million [9] and $35 billion [10-11], respectively. The
57 prominent bacteria responsible for causing mastitis include both gram-positive and gram-
58 negative bacteria like staphylococci, streptococci, Escherichia coli and Klebsiella pneumoniae
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59 (12). Coagulase-negative staphylococci (CNS) are opportunistic pathogens that have also
60 emerged as important mastitis pathogens (13). Antibiotics are used for the treatment of bovine
61 mastitis, but these are associated with the problem of antibiotic resistance. The prevalence of
62 mastitis pathogens and their antimicrobial resistance have been investigated in numerous studies
63 around the world. For example, studies from Ethiopia and Estonia have reported a high

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64 prevalence of penicillin resistant S. aureus and CNS strains in these countries [14, 15]. Similarly,
65 in West Bengal, India, Gram-negative bacteria have been recently found to be resistant to

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66 antibiotics like β-lactams and tetracyclines [16]. In Central Mexico, antimicrobial investigations
67 revealed that the isolated mastitis pathogens were resistant to penicillin, clindamycin and

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68 cefotaxime [17]. Antimicrobial studies from Canada have also exhibited that subclinical isolates

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69 of two important mastitis pathogens, Streptococcus uberis and Streptococcus dysgalactiae
70 harbour antimicrobial resistance genes and should be considered as potential propagators of
71 antimicrobial resistance [18]. In Southern Taiwan, E. coli isolates from milk samples of mastitis
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72 affected cows were fluoroquinolone-resistant and extended-spectrum β-lactamase-producing
73 strains [19]. Therefore, there is a need to explore alternative approaches for the treatment of
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74 mastitis. This review article is an overview of the different medicinal plants used in the treatment
75 of bovine mastitis and highlights their importance as an alternative natural product therapeutic
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76 resource.
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77 1.2. Bioprospecting medicinal plants

78 Bioprospecting is the discovery of natural products that have a useful pharmacological or

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79 biological application [20]. In many cases, bioprospecting involves the search for useful organic
80 compounds in natural products like microorganisms, plants, and fungi that grow in extreme
81 environments, such as rainforests, deserts, and hot springs [21]. Natural products traditionally
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82 acted as primary source for more than 80% of the drug substances and even today hold an
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83 important place in the drug discovery process [22, 23]. A data analysis of the literature available
84 from 1981 – 2006 showed that more than 50 percent of the drugs approved by FDA were natural
85 products or natural products derivatives [24]. Natural products have proved to be the primary
86 source for antibacterial drug discovery also. About 66% of all the drugs currently approved as
87 antibacterial agents are natural products or natural product derivatives [25].
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88 Pressure to find novel antibacterial products with new modes of action that target non-essential
89 cell processes has driven exploitation of plant sources for the identification of new and effective
90 antibacterial agents [26]. The mechanisms of action of phytochemicals may be different from
91 those of routine antibiotics and this feature could be of vital importance in the treatment of
92 resistant bacteria. Combining phytochemical antibacterials with already existing drugs offers

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93 another field for application of phytochemicals and should be pursued extensively. While there
94 are no plant-derived single chemical entity antibacterial drugs available on the market yet, this

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95 natural source is worth exploration due to several important reasons as has been excellently
96 reviewed by several authors [27-30]. Plants can serve as a novel and alternative source of

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97 antibiotics [31]. Several phytochemicals, either with promising antibacterial activity such as

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98 horminone, pyrithione, gossypol, plumbagin and rhein, or as efflux pump inhibitors like 5'-
99 methoxyhydnocarpin (5'-MHC) have been isolated from different plant species [32-33]. Plant
100 antibacterial agents can also target bacterial virulence and this class of antibacterial agents
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101 remains largely untapped [34].

102 Antibiotic resistance can also develop against plant based antibacterial agents. However, during
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103 the course of evolution, plants may have evolved some different chemical strategy for the control
104 of microbial infections in order to decrease the selective pressure for developing antibiotic
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105 resistance. For example, plant antibacterials usually act in combinations and have little efficacy
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106 alone [34].

107 Medicinal plants used against mastitis: In organic dairy farming, farmers replace antimicrobials
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108 with non-traditional herbal therapies. Herbal therapies are an essential component of
109 ethnoveterinary medicine (EVM). EVM is particularly important in animal health care in
110 developing countries [35]. It has become a recognized field of research that includes traditional
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111 veterinary theory, medicines, surgical methods, diagnostic procedures and animal husbandry
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112 practices [36]. Farmers and pastoralists in several countries use medicinal plants in the
113 maintenance and conservation of the healthcare of livestock. Approximately, 75% of rural
114 livestock owners in the Eastern Cape province of South Africa use plants or plant-based
115 remedies to treat their livestock [37]. In India, EVM is used for primary health care treatment to
116 make domestic animals productive and healthy [38]. The indigenous knowledge of the veterinary
117 health care system acquired by traditional herbal healers is orally transformed from one
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118 generation to the other. Herbal therapy is also used in the treatment of mastitis in different parts
119 of the world. For example, in the United States, organic farmers treat clinical mastitis using a
120 variety of alternative therapies including whey based products, botanicals, vitamin supplements,
121 and homeopathy [39]. Similarly, a number of plant species like Spathodea campanulata and
122 Tridax procumbens are used to cure mastitis in India [40]. In British Columbia, Canada, mastitis

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123 is treated with Achillea millefolium, Arctium lappa, Salix alba, Teucrium scorodonia and Galium
124 aparine [41]. Organic dairy farmers in different parts of the world use a variety of nontraditional

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125 methods to cure mastitis. Medicinal plants used against bovine mastitis may have different
126 biological properties. However, there is limited efficacy data to support the use of these

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127 alternative treatments. Therefore, it is important to scientifically validate different traditional

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128 plant remedies. Although different alternative treatment approaches are used in the control of
129 mastitis, the goal here is to highlight only those medicinal plants which have been investigated
130 for antimicrobial potential against mastitis pathogens.
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131 1.3. Antimicrobial studies of plants or plant derived molecules against
132 mastitis: Although plants are widely used against different animal diseases including bovine
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133 mastitis, there are limited reports available on the pharmacological study of medicinal plants
134 used in the control of mastitis. Below we present a brief compilation of some of the literature
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135 reports available on the exploration of medicinal plants for their potential use against bovine
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136 mastitis.

137 Hase et al., evaluated and compared the therapeutic efficacy of topical herbal spray
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138 (AV/AMS/15) with Mastilep gel against subclinical mastitis [42]. The phytochemical
139 constituents of plant species Cedrus deodara, Curcuma longa, Glycyrrhiza glabra and
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140 Eucalyptus globulus are used in alternative medicine for their antibacterial, anti-inflammatory,
141 analgesic, anti-histaminic and immunomodulatory properties. These herbal ingredients at various
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142 concentrations form the basis of Mastilep gel and herbal spray (AV/AMS/15). For experimental
143 purposes, cows detected with subclinical mastitis were divided into three groups: one control
144 group and two treatments. Group A i.e., control group was not given any treatment. However,
145 first and second treatment groups i.e., Group B and Group C were treated with herbal spray
146 (AV/AMS/15) and topical application of Mastilep gel, respectively. Both the herbal formulations
147 were sprayed and gently applied to the cow udders twice a day after every milking for five
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148 consecutive days. The authors claimed that use of this herbal spray on test animals led to an
149 improvement in milk yield and milk fat content and reduced SCC by boosting the udder
150 immunity in comparison to the control group. Thus the herbal spray not only eliminated the
151 udder infection in sub clinical mastitis but also helped in the control of mastitis without any side
152 effects. The study proved that the overall cure rate and prophylactic efficacy of AV/AMS/15

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153 topical herbal spray was good and at par to Mastilep gel.

154 Since mastitis reduces milk yield and significantly alters milk composition depending on the

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155 severity and duration of the infection and the pathogens involved, therefore, changes in milk
156 composition are used to assess udder health. Generally, mastitis is characterized by an increase in

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157 the blood constituents in milk and a decrease of normal milk constituents. While assessing such

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158 effects, Sunder et al., 2013 analyzed the effect of Morinda citrifolia fruit juice on milk
159 characteristics of 13 healthy and 12 mastitis-affected dairy cows. The fruit juice was fed orally to
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160 the animals at the rate of 100 ml/day/animal. The authors noticed that the administration of the
161 fruit juice significantly lowered the pH of mastitis affected milk from 7.2 ± 0.17 to 6.54 ± 0.07.
162 The electrical conductivity of mastitis-affected milk also lowered significantly from 5.97 ± 0.81
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163 × 105 mS/cm to 4.77 ± 0.14 × 105 mS/cm. It was further observed that the fruit juice reduced the
164 total bacterial count in the mastitis milk to a significant level from 5.15 ± 0.03 × 108 to 2.54 ±
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165 0.03 × 108 CFU/ml. The fruit juice feeding also decreased the total protein concentration in the
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166 mastitis milk from 41.83 ± 6.56 µg/ml to 21.13 ± 0.93 µg/ml. No significant change for these
167 parameters was observed in the treated group of healthy animals. Although, the milk yield was
168 not significantly changed in both the treatment groups but the mastitis affected animals showed
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169 some increase in milk yield after feeding of the fruit juice. Therefore, the overall results showed
170 that feeding of M. citrifolia fruit juice to mastitis affected cows resulted in the positive change in
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171 the milk composition and thereby improving the udder health [43].
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172 Ocimum sanctum (Tulsi) is an important plant species used in the treatment of various animal
173 diseases. It possesses immuno-modulatory and anti-inflammatory properties attributed to its
174 active constituents such as volatile oil (eugenol, 80%), flavonoids, and triterpene. Shafi et al.,
175 2016 evaluated the immunotherapeutic potential of this important medicinal herb against mastitis
176 affected dairy cows [44]. Twenty Holstein Friesian × Sahiwal lactating dairy cows were selected
177 for this study. The cows were divided into two equal groups: a control group and a group orally
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178 administered the leaf powder of tulsi at a concentration of 600 mg/kg body weight on a daily
179 basis for 7 days. It was found that the treatment reduced 69.23% of intramammary infections and
180 also resulted in a significant reduction in somatic cell count and ceruloplasmin concentration,
181 therefore decreasing udder inflammation and improving milk quality. The significant increase in
182 phagocytic activity of milk neutrophils and enhanced lactoperoxidase and myeloperoxidase

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183 activities proved that the herb possesses immune-modulatory potential. Therefore, the study
184 proved the immunotherapeutic potential of tulsi in the treatment of subclinical mastitis.

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185 Reshi et al., 2017 demonstrated the antibacterial activity of Fumaria indica and Adiantum
186 capillus against subclinical mastitis by intra-mammary infusion of these plant extracts [45]. This

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187 was done after preliminary screening of extracts from three plant species for their safety by

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188 carrying out cytotoxicity studies on HeLa cell line using MTT cell proliferation assay. The plant
189 extracts were then assessed for their antibacterial activities against S. aureus, E. coli, S.
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190 agalactiae and K. pneumoniae by using disc diffusion method. It was found that Fumaria indica
191 extract showed best antibacterial activity followed by Nepata cataria and Adiantum capillus. In
192 order to further check their efficacy, in vivo studies were carried by intramammary infusion of
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193 aqueous extracts at a dosage of 750 mg/tube for 5 days in cows affected with subclinical mastitis.
194 For this purpose, animals were placed into five groups: Group II, Group III and Group IV were
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195 treated with aqueous extracts of Fumaria indica, Adiantum capillus and Napeta cataria,
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196 respectively. Groups I and V served as negative and positive controls. Recovery was confirmed
197 by correlating the California Mastitis Test score on day 5 with somatic cell count (SCC) as
198 compared with day zero of therapy. Highest clinical recovery was reported in case of Fumaria
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199 indica followed by Adiantum capillus. Therefore, herbal preparations of Fumaria indica and
200 Adiantum capillus can be considered as an alternative treatment for mastitis.
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201 A topical polyherbal formula known as Ya-Sa-Marn-Phlae (YSMP) has been traditionally used
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202 in Thailand for the treatment of wounds and inflammatory skin diseases. It consists of Curcuma
203 longa (rhizome), Areca catechu (seed), Oryza sativa (seed), and Garcinia mangostana
204 (pericarp). Chusri et al., evaluated the antibacterial potential of this polyherbal formula, its
205 herbal components (Curcuma longa, Areca catechu, Oryza sativa, and Garcinia mangostana),
206 and representative chemical constituents (catechin, α-mangostin, and curcumins) against both
207 coagulase positive and coagulase negative staphylococci isolates [46]. The results showed that
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208 ethanol extracts of polyherbal formula, G. mangostana, and α-mangostin showed most promising
209 antibacterial activities with MIC values of 1–32 µg/ml. When the test samples were investigated
210 for their anti-biofilm activity, it was found that these significantly inhibited biofilm formation of
211 the test isolates. Further studies revealed that the polyherbal formula showed similar results like
212 that of α-mangostin and G. mangostana. Electron microscopic studies showed that the

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213 polyherbal formula and G. mangostana act by causing severe alterations in the bacterial cell wall
214 by the formation of holes and morphological disorganization. The studies also proved that G.

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215 mangostana is the most active component of the YSMP. However further investigation are
216 needed to correlate the activity of either YSMP, G. mangostana, or their constituents.

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217 While evaluating the antimicrobial potential of an indigenous polyherbal preparation on

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218 periparturient immunity and udder health of high yielding Karan-Fries crossbred cows, Sharma
219 et al., 2014 noticed that the herbal preparation showed promising results [47]. The herbal product
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220 consists of Withania somnifera, Asparagus racemosus, Emblica officinalis, Ocimum sanctum,
221 Tinospora cordifolia, Tribulus terrestris, and Nigella sativa. The preparation was given to the
222 cows at the rate of 200–250 mg/kg body weight. It was observed that test product significantly
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223 reduced periparturient stress while enhancing immunity and improving the udder health in test
224 animals. These primary studies suggest that the herbal product can be probably used as feed
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225 additive to reduce the incidence of mastitis.

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226 Leaves form an integral component of most natural pastures for ruminant diets and chemical
227 investigations have shown that tree leaf components like tannins, essential oils, or other aromatic
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228 compounds have anti-nutritional or anti-microbial factors. Based on this hypothesis, researchers
229 evaluated the preliminary antimicrobial activity of tannin extracts from eight plant species
230 common to the Southern Great Plains of USA [48]. The evaluated plant species showed a dose
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231 dependent in vitro inhibition of S. aureus, in the following order: Shinnery oak > Post oak >
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232 Locust > Blackjack oak ≥ Skunk bush > Sericea lespedeza > commercial Quebracho ≥ Sumac >
233 Plum. The extracts from two plant species i.e., Shinnery and Post oaks showed maximum growth
234 inhibition of S. aureus. As the tannin extracts from leaves of these plant species were highly
235 inhibitory to selected pathogens, the researchers recommended that these may provide
236 alternatives and supplements to conventional antimicrobial feed additives.
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237 Different medicinal plants Symphythum officinale, Sambucus nigra, Mentha sp., Ocimum
238 basilicum, Parapiptadenia rigida, Cuphea carthagenensis, Salmonella cholleraesuis,
239 Alternanthera brasiliana, Achillea millefolium, Baccharis trimera and Solidago chilensis are
240 used in the prevention and control of bovine mastitis in Southern Brazil [49]. Decoctions
241 prepared from these plants were analyzed for their in vitro antimicrobial activity against S.aureus

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242 and Salmonella choleraesuis by the agar dilution method in order to scientifically validate the
243 traditional practice. Extracts from Alternanthera brasiliana, Achillea millefolium, Baccharis

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244 trimera and Solidago chilensis inhibited the growth of S. aureus while Symphythum officinale,
245 Sambucus nigra, Mentha sp., Ocimum basilicum, Parapiptadenia rigida and Cuphea

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246 carthagenensis extracts were active against both microorganisms. The results thus justified to

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247 some extent the traditional use of the above plant species against bovine mastitis.

248 As part of the search for alternative therapeutic approaches to antibiotics for the treatment of
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249 mastitis, the efficacy of short chain fatty acids viz., caprylic acid and monocaprylin were
250 examined for their ability to inhibit the growth of five common mastitis pathogens, i.e., S.
251 agalactiae, S. dysgalactiae, S. uberis, S. aureus, and E. coli. The antimicrobial studies showed
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252 that both caprylic acid and monocaprylin were bactericidal against the above microorganims at
253 the tested concentrations and reduced the growth of all pathogens by >5.0 log CFU/ml after 6 h
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254 of incubation [50]. The streptococci species were found to be most sensitive while E. coli was
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255 most tolerant to the test samples. The results indicate that both caprylic acid and monocaprylin
256 should be further evaluated as alternatives or adjuncts to antibiotics as intramammary infusion
257 strategies to treat bovine mastitis.
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258 In another similar study, a research group explored the antimicrobial potential of four essential
259 oil components Trans-cinnamaldehyde (TC), eugenol, carvacrol, and thymol against mastitis
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260 pathogens inoculated in milk samples [51]. These four essential components have been classified
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261 as GRAS (generally regarded as safe) by the United States Food and Drug Administration. The
262 test antimicrobials were evaluated for minimum inhibitory concentration (MIC) and minimum
263 bactericidal concentration (MBC) determination against major mastitis pathogens like S.
264 agalactiae, S. dysgalactiae, S. uberis, S. aureus, and E. coli. The four molecules inhibited the
265 growth of test pathogens but TC was most effective in reducing the bacterial growth. In time-kill
266 assays, TC reduced the bacterial load in milk by 4.0 to 5.0 log CFU/ml and to undetectable levels
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267 within 12 and 24 h, respectively. Further, the antimicrobial effect of TC persisted for the duration
268 of the experiment (14 d) without any loss of activity. These results suggest that among the test
269 molecules, TC has the potential to be further evaluated as an alternative or adjunct to antibiotics
270 as intramammary infusion to treat bovine mastitis.

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271 Similarly, a research team evaluated the antibacterial activity of three plant-derived diterpenes
272 against a panel of microorganisms known to cause bovine mastitis [52]. From the three evaluated
273 diterpenes, ent-Copalic acid (CA) was found to be the most active metabolite, with promising

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274 MIC values (from 1.56 to 6.25 µg ml) against S. aureus, S. epidermidis, S. agalactiae, and S.
275 dysgalactiae. Time-kill assays of CA against S. aureus showed that it has a bactericidal effect.

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276 Therefore, the study concluded that CA could be of interest in the control of several Gram-

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277 positive bacteria associated with bovine mastitis.

278 While assessing the antimicrobial activities of leaves of Spathodea campanulata and Tridax
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279 procumbens, Das et al., found that the methanol extract of S. campanulata inhibited the growth
280 of S. agalactiae, S. uberis, E. coli and S. aureus [53]. However, the methanol extract of T.
procumbens was only active against S. aureus. Further, phytochemical screening of the plant
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281
282 species revealed the presence of alkaloids, tannins, saponins, steroids, terpenoids and flavonoids.
283 As tannins are well known antimicrobial agents and tannin-containing plants are used to treat
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284 diarrhoea, inflammatory disorders and skin problems, the research team used these points as the
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285 rationale behind using these plants as a natural remedy to cure mastitis in dairy cows.

286 In search of natural alternatives for the treatment of mastitis, Montironi and coworkers [54]
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287 evaluated the antimicrobial efficacy of Minthostachys verticillata essential oil and one of its
288 major constituents, limonene against S. uberis strains isolated from bovine mastitis. The MIC
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289 and MBC values ranged from 14.3 to 114.5 mg/ml and 114.5 and 229 mg/ml in case of the
290 essential oil while the values for limonene were found to be 3.3 to 52.5 mg/ml and 210 mg/ml
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291 respectively against the different tested strains of S. uberis. Since S. uberis strains are considered
292 to be strong biofilm formers, the test samples were also evaluated for their anti-biofilm activity
293 and it was found that both these agents reduced biofilm formation. The biofilm inhibition of the
294 essential oil MIC ranged from 88.25 ± 7.62 % to 23.50 ± 16.26 % while that of limonene from
295 92.18 ± 4.78 % to 23.20 ± 16.05 %. As Minthostachys verticillata essential oil and its major
296 constituents have shown no toxic effects in both in vitro and in vivo assays, therefore the authors
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297 argue that these could be used as an alternative and/or complementary therapy for bovine
298 mastitis caused by S. uberis in future after further evaluation studies.

299 The leaf extracts of Liquidambar orientalis (L. orientalis) were evaluated for their antibacterial
300 activity against different S. aureus strains and CNS responsible for causing subclinical mastitis in

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301 cows [55]. In addition, the extracts were also checked for their antioxidant potential. The results
302 showed that acetone extract had best antibacterial activity against S. aureus isolate numbered -17
303 with zone of inhibition equal to 12 mm. However, all the three solvent extracts i.e., acetone,

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304 methanol and ethanol showed best MIC value of 3.2mg/ml against the tested bacterial strains.
305 Anti-oxidant activity was determined by ABTS [2, 2'-azino-bis (3-ethylbenzothiazoline-6-

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306 sulphonic acid)] free radical assay where ethanol extract exhibited a strong anti-oxidant activity.

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307 Alves et al., 2016 reviewed some of the medicinal plants explored for their antimicrobial activity
308 against mastitis pathogens. They observed that the antimicrobial potential of essential oils of
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309 plant species like oregano (Origanum vulgare), Mexican oregano (Lippia graveolens), thyme
310 (Thymus vulgaris) and that of constituents like carvacrol, thymol and cinnamaldehyde are of
importance [56].
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312 Gomes and Henriques [57] provided an overview of some of the conventional and emerging
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313 approaches in the management of bovine mastitis infections. The list included bacteriophages,
314 vaccines, nanoparticles, cytokines, and natural compounds from plants, animals, and bacteria as
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315 some of the valid substitutes to antibiotics. The review discusses about the positive results of
316 plant based antimicrobials on important mastitis pathogens and suggests their potential use as an
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317 alternative treatment source to antibiotics.

318 Pasca et al., 2017 investigated the antimicrobial efficacy of alcoholic extracts from eleven plant
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319 species and eight plant-derived products against a panel of 32 test microorganisms isolated from
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320 milk samples [58]. The eight herbal products were produced due to a combination of various
321 plant extracts in different concentrations. The results showed that three plant species viz.,
322 Evernia prunastri, Artemisia absinthium, and Lavandula angustifolia inhibited the growth of test
323 microorganisms to the maximum level. Similarly, from the eight evaluated plant products, only
324 three samples R3, R4 and R7 showed best antimicrobial activity comparable to the standard
325 antimicrobial drugs florfenicol and enrofloxacin. The plant products showed better activity than
326 individual plant extracts possibly due to synergistic effect of the plant products.
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327 1.4. Our studies: In recent years, our research group has been working on the exploration of
328 traditionally used medicinal plants of Kashmir Himalayas, India, in the control of bovine
329 mastitis. During these preliminary studies, we have evaluated some medicinal plants for their
330 antimicrobial potential against important mastitis pathogens. For example, while working on one
plant species viz., Aquilegia fragrans, the crude methanol extract of the underground parts along

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332 with five isolated constituents exhibited moderate to weak antibacterial activities against mastitis
333 pathogens [59]. Similarly, another plant species Thalictrum minus was pursued for alkaloid

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334 isolation because alkaloids are a large and structurally diverse group of compounds that serve as
335 scaffolds for important antibacterial drugs such as metronidazole and the quinolones [60]. We

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336 isolated three bisbenzylisoquinoline alkaloids i.e.; Thalrugosaminine, O-Methylthalicberine and

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337 5'-Hydroxythalidasine from Thalictrum minus that inhibited the growth of important mastitis
338 pathogens [61].
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339 1.5. Future scope: Extensive bioprospecting studies are needed in this direction to discover
340 the tremendous medicinal potential of various plant species from biodiversity rich areas of the
world. The medicinal plants used as an alternative therapeutic option against bovine mastitis can
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342 act as antibacterial, anti-inflammatory or immune-modulatory agents which need to be explored.
343 As considerable oxidative damage occurs in the mammary glands during bacterial infection in
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344 mastitis, there are also likely opportunities for pharmacological intervention to block this
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345 proteolytic or oxidative cascade. Since plants are an excellent source of antioxidants, the use of
346 plant antioxidants to prevent this oxidative stress in bovine mastitis offers another opportunity
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347 for exploration of medicinal plants in the control of this chronic disease.

348 Acknowledgements: The first author is thankful to University Grants Commission, New
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349 Delhi India for fellowship support.

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350 REFERENCES
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• Bovine mastitis is the most common and costly disease affecting dairy herds.

• Antibacterial agents are associated with the problem of antibiotic resistance.

• Herbal cure is an alternative therapeutic option for the treatment of bovine mastitis.

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• An overview of different medicinal plants used against mastitis is presented.

• It highlights the logic and precedence behind mining this natural product resource.

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