S. Amsaveni, S. Pavulraj, D. Silambarasan, K. Saranya and S.

Ramesh

Department of Veterinary Pharmacology and Toxicology,

Madras Veterinary College, Chennai 600 007
Mastitis is one of the most important diseases of
high yielding dairy animals.

Fungal mastitis -2-3% of clinical cases seen in veterinary

practice

Though there are potent antifungal drugs

they are not used in the treatment of fungal mastitis

Plants with antifungal properties can be exploited to

provide safe and cost effective treatment
 Mangifera indica Linn (Mango seed
kernel)

Aqueous
Aqueous
Terminalia chebula

Plants selected in this study

Aqueous

Psidium gujava Linn (guava leaves)

Aqueous
Punica granatum Linn( pericarp)

Vitis vinifera
(seeds)

Alcoholic extract
Agar well diffusion assay -Hassawi and Kharma(2006) with slight modifications

Molten SDA

Cool
5 x106CFU/ml

Candida spp
Punch wells
Add the extract of
known
concentration to
each well

Incubate at 37ºC,24-48hrs

+ control - Amphotericin B
Measure the zone of inhibition -control- 5%DMSO
DIGITAL CALIPERS
 Agar well diffusion assay for T.chebula

28
26
24
(Zone diameter mm)

22
20
18
16
14
12
10
8
6
4
2
0

C12 801 R 783 800 845

Isolate numbers /concentration mg/ml

150mg/ml 300mg/ml 500mg/ml

 Agar well diffusion assay for
Vitis vinifera seed extract

20
Zone of inhibition(mm)

18
16
14
12
10
8
6
4
2
0
C12 801 R 783 800 845
Isolates

150mg/ml 300mg/ml 500mg/ml

 Agar well diffusion assay for
Mango seed kernel extract
20

18

16
Zone of inhibition(m m )

14

12

10

4 Agar well diffusion assay for Amphotericin B

35 10/μg/ml
2
30
0

C12 R 800 inhibition(mm) 25

Zone of 20
Isolate 15

200mg/ml 500mg/ml
10

R 800 801 845 783 C12

 Agar well diffusion assay for Guava leaf
extract and Pomegranate pericarp extract
18
16
in h ib itio n (m m )

14
12
Zone of

10
8
6
4
2
0

845 845
concentration of the extract(mg/ml )

10mg/ml 200mg/ml 10mg/ml 100mg/ml 200mg/ml

 Grape seed extract –Isolate 800

300mg/ml

5%DMSO
300mg/ml
300mg/ml
150mg/ml
150mg/ml
500mg/ml

5%DMSO

150m
Grape seed extract –Isolate C12
500mg/ml 500mg/ml

lg/m
T.Chebula isolate 801

300mg/ml

500mg/ml
150mg/ml
 T. chebula Isolate 801

300mg/ml

500mg/ml 150mg/ml

500mg/ml
Amphotericin B
10μg/ml
200mg/ml
Mangifera indica
Mangifera indica 500mg/ml

Isolate C12

Amphotericin B
10μg/ml Pomegranate

200mg/ml
200mg/ml

Isolate 845
 Resazurin dye microbroth dilution method
(Sarkar et al.,2007)

5 x106CFU/ml
•100μl extract in 1st well
•50 μl sterile nutrient broth in 2-12th well
•Serial two fold dilution
96 WELL MICROTITRE PLATE
•10 μl dye and 30 μl sterile broth from 1-
12 wells
•Finally add 10 μl fungal
Highest dilution of suspension(5x106CFU/ml)
the extract •Loosely wrap and incubate at 37ºC for
showing inhibition 24-48hrs.
of fungal growth.
•Fungal growth-pink color
(The well before
Growth inhibition- blue color
pink color change
taken as MIC of
that extract
 MIC Results
Sl.no Extract MIC value(mg/ml)

1 Mangifera indica Linn (Mango 0.625

seed kernel)
2 Psidium gujava Linn (guava 2.5
leaves)
3 Punica granatum Linn( pericarp) 5
4 Terminalia chebula 0.3125-0.625
5 Vitis vinifera (seed) extract 0.625-0.078
Amphotericin B 10µg/ml 0.625µg/ml
MIC grape seed extract

0.078mg/ml

0.156mg/ml

0.156mg/ml

0.625mg/ml

0.078mg/ml

0.625µg/ml
MIC results of T.chebula

0.3125mg/ml

0.3125mg/ml

0.625mg/ml

0.625mg/ml

0.625µg/ml
• All the extracts tested in this study posses
activity against yeast (candida spp) isolated from
bovine mastitis.
• Grape seed extract said to possess broad spectrum antibiotic
effect due to the presence of natural quaternary compound and
used in vaginal yeast infections in women as douche. (Dr.Leo
galland).
• T. chebula contains tannins, anthraquinone, chebulinic acid,
chebulegic acid ,shown to reduce growth of C. albicans.
(Elizabeth et al.,2005) .
• Gallotannins of Mangifera indica seed kernel shown to possess
activity against Bacillus subtilis (Ganzle and Schieber, 2010).
• Punica granatum Linn (Pomegranate) might be used as an
effective anti-bacterial agent against oral biofilm bacteria
(Pereira et al., 2006) they have shown to contain tannins.
• Antibacterial activity of guava leaves against mastitis causing
bacteria was studied by (Inchaisri et al.,2005).Guava leaf is rich
in flavanoids whose antibacterial activity was demonstrated by
several researchers (Abdelrahim et al.,2002).
 Conclusion

This property can be exploited for

developing herbal formulations for mastitis
treatment.