Imtiyaz Ahmad Reshi1, Tarun Kumar Sarkar1, Ashiq Hussain Bhat2H.U. Malik1, Amatul Muhee1 and Sabia Qureshi3

1Department of Clinical Veterinary Medicine, Ethics and Jurisprudence,
Faculty of Veterinary & Animal Science, SKUAST-K J&K, India.
2Division of Veterinary Gynaecology and Obstetrics,
Khalsa college of Veterinary and Animal Sciences, Amritsar, 143001, India.
3Division of Veterinary Microbiology and Immunology, Faculty of Veterinary Sciences and Animal Husbandry, Sher-e-Kashmir University of Agricultural Sciences & Technology, Kashmir, J&K -190006, India.

Abstract

The study was conducted to find out the activity of some medicinal plants against bacterial isolates. The bacterial isolates from milk samples of infected quarters of cows were investigated for in-vitro drug sensitivity by standard disc diffusion technique (Bauer et al., 1966). Cultural examination of milk samples was done by method described by Quin et al. (2004) in which predominant isolates were Staph.  (55.55%), E. coli (22.22%), Strepto. agalactiae (16.66%) and other isolates as 5.5% and were subjected to in-vitro antibacterial sensitivity test to selected herbal extracts and standard antibiotic (cefuroxime) (Table-1). The four different aqueous concentrations of the herbs namely Fumaria indica, Adiantum capillus, Nepata cataria, Levandula stoeches and Borago officinalis collected from registered herbal shops prepared by standard procedure as 25mg/ml, 50mg/ml, 75mg/ml and 100mg/ml were used. The results indicated that aqueous extract of Fumaria indica, Adiantum capillus and Napeta cataria against Staphylococcus aureus, E.coli, Streptococcus agalactia and K. pneumonia exhibited maximum zone of inhibition 20.0±1.21, 21.0±0.19, 13.0±0.37 17.0±0.21; 18±0.41, 12±0.21, 13±0.31, 15±0.31 and 15±0.33, 16.01±0.19 14.09±0.37, 13.31±0.41 at 100 mg/ml respectively which was significantly low as compared to standard drug (cefuroxime) at 30 microgram concentration. Aqueous extract of Levandula stoeches against Staphylococcus aureus and E.coli exhibited maximum zone of inhibition 18.0±0.33 and 13.0±0.141 at 100 mg/ml respectively. Borago officinalis has shown nil to non-significant bacterial growth inhibition activity.

Keywords: Antibacterial, herbs, aqueous extract, zone of inhibition.

Introduction

Plants being a source of many potent and powerful drugs are used medicinally in different countries (Srivastava et al., 1996). A wide range of medicinal plant parts like roots, stems, flowers, fruits, twigs, exudates and modified plant organs are used for extract of raw drugs and they possess varied medicinal properties. Considering the vast potentiality of plants as source for antimicrobial drugs the present study was undertaken to screen the antibacterial potential of aqueous extracts of some plants of Kashmir, J&K, India.

1) Nepeta cataria: Nepeta, a genus of annual or perennial herbs, belonging to the Lamiaceae family, includes approximately 250 species and are localized in central and southern Europe, Asia, the Middle East, northern Africa and tropical mountains in Africa (Ghannadi et al, 2003). Nepeta species are used as the traditional medicine in many countries and have a large ethno-botanical effects like diuretic, diaphoretic, vulnerary, antitussive, antispasmodic, antiasthmatic, tonic, febrifuge and carminative (Ghannadi et al., 2003). The present study was aimed to evaluate the antimicrobial potential of the Nepeta species found in Kashmir.

2) Fumaria indica/officinalis: The genus Fumaria (Fumariaceae) consists of 46 species in the world and are known as “fumitory, earth smoke, beggary, fumus, vapor, fumittery or wax dolls” in English. Fumaria officinalis is a small, branched, annual herb growing wild in plains and lower hills and is locally known as “Pitpapra” or “Shahtrah” in India “Shahterah” in Kashmir. Literature reveals that the extracts of Fumaria indica posses spasmolytic, analgesic, anti-inflamatory, antibacterial properties.

3) Borago officinalis: This plant in India is sparsly distributed in Northen-Eastern Himalayas from Kashmir to Kumaon at altitudes of 3,500-4,500m ASL and occurs during November to January.

4) Adiantum capillus: It has a worldwide distribution, found in Pak-Indian subcontinent, western Himalaya, Mexico, warmer parts of America and other tropical and subtropical regions of the world (Nisar et al., 2012; Reddy, 2010). Adiantum is used as expectorant, astringent, demulcent, antitussive, emmenagogue, febrifuge, diuretic and in catarrhal affections.

 

5) Levandula stoeches: The lavenders are a genus of about 25-30 species of flowering plants in the mint family, Lamiaceae, native of Mediterranean region south to tropical Africa and many regions of Asia and has been used for centuries as a herbal remedy for many ailments. Lavender yields highly effective essential oil with very sweet overtones which is believed to be of benefit for a multitude of problems including stress, anxiety, exhaustion, irritability, head ache, migraine, insomnia, depression, cold, indigestion, liver, gall bladder problems and cancer (Hudson 1996; Henley et al. 2007). Reports of antibacterial effects have also been documented.

 Material methods:

Following herbs (aqueous extract) were evaluated for their in vitro toxicity trial on cell line (HeLA cell lines):

S. No.
Name of the herb
Local name
Part used
1
Nepata cataria
Gandh soi
Leaves
2
Levandula stoeches
Kalwuth
Leaves
3
Fumaria indica
Shahter
Leaves
4
Adianthum capillus
Gavtheer
Leaves
5
Borago officinalis
Kahzaban
Leaves

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 extracts of herbals were prepared. The test microorganisms were seeded into respective media by spread plate method with the 24 h cultures of bacterial growth in nutrient broth. After solidification the filter paper discs (5 mm in diameter) impregnated with the extracts of different concentrations were placed on test organism-seeded plates. S. , E. coli and other isolates were used for antibacterial test. The antibacterial assay plates were incubated at 37°C for 24h and the diameters of the inhibition zones was measured in mm, Cefuroxime was used as positive control.

In vitro antibacterial sensitivity test of herbs in comparison with standard antibiotic

The bacterial isolates from milk samples of infected quarter were further investigated for in vitro drug sensitivity by standard disc diffusion technique (Bauer et al., 1966) using commercially available selective antibiotic discs (M/S Hi-Media Laboratories).

Antibiogram

The different concentrations of herbs were studied for in vitro AST in comparison to standard antibiotic. The diameter of the zone of inhibition was measured by paper scale after 18 hr incubation for identifying the sensitivity of microbial culture against above mentioned extracts according to laboratory manual.

Cultural examination of milk

Cultural examination of milk samples was done by method described by Quin et al. (2004). Milk samples collected in sterile glass vials were streaked primarily on ovine blood agar plates with a sterile platinum loop under strict sterile environment. The inoculated plates were incubated at 37oC for 24 hours. The causative organisms were identified initially by colony characteristics on blood agar, Gram staining and biochemical characteristics for presence of catalase and cytochrome C oxidase. Further, the organisms grown on blood agar plates were streaked on selective media e.g. Mannitol Salt Agar (for Staphylococcus spp.), Edward’s media (for Streptococci), MacConkey Agar (for Coliforms and Enterococci). Hotis test was done to identify Streptococcus agalactiae.

 Result and discussion:

Bacterial isolation

The predominant isolates were Staph.  (55.55%), E. coli (22.22%), Strepto. agalactiae (16.66%) and other isolates as 5.5%. Thus Staph. , E. coli, K. pneumonia and Strepto. agalactiae were selected for in vitro antibacterial sensitivity test to selected herbal extracts. Among all the pathogens of bovine mastitis, Staphylococcus  is the predominant organism (Allore, 1993; Kapur et al., 1992). In India, previous and several studies about bovine mastitis (1994; Costa et al., 1997; Naiknaware et al., 1998) have assessed that the coagulase negative Staphylococci were the most frequent isolated bacteria

In vitro antibacterial sensitivity test of herbs in comparison to standard antibiotic

            Four different concentrations of herbs were studied for in vitro AST in comparison with standard antibiotic.           

Fumaria indica

Aqueous extract of Fumaria indica against Staphylococcus aureus and E. coli exhibited maximum zone of inhibition 20.0±1.21 and 21.0±0.19 at 100 mg/ml and minimum zone of inhibition 9.0±0.51 and 11.0±0.22 was recorded at 25 mg/ml respectively which significantly increased at 50 and 75 mg/ml where zone of inhibition was 11.0±0.33 and 17.0±0.19, 13.0±0.41 and 19.0±0.16 respectively. Against Streptococcus agalactia exhibited maximum zone of inhibition 13.0±0.37 was recorded at 100 mg/ml and minimum zone of inhibition (1.0±0.12) was recorded at 25 mg/ml which significantly increased at 50 and 75 mg/ml where zone of inhibition were 6.00±0.41 and 7.0±0.04, respectively while as, against K. pneumonia, maximum zone of inhibition 17.0±0.21 was reported at 100 mg/ml and minimum zone of inhibition (4.0±0.90) was recorded at 25 mg/ml which significantly increased at 50 and 75 mg/ml where zone of inhibition were 9.0±0.19 and 12.0±0.57, respectively. Aqueous extract of Fumaria indica exhibited high antibacterial activity against Staphylococcus aureus, E. coli, K. pneumonia and and the findings match with Khan et al. (2014), Gupta et al. (2012), Fatima et al. (2014). Parekh and Chanda (2007) and Fazal et al. (2012) and the findings are in contrast to Shinwari et al. (2015). The antimicrobial activity of aqueous extract of Fumaria indica against the different clinical strains of bacteria supported the scientific validity of the plant being used traditionally as a medicine. The inhibition of bacterial strains by aqueous extract may be attributed to the presence of soluble phenolic and polyphenolic compounds in the extract. The significant antimicrobial effects of this extract could be explained by disturbance of permeability barrier of bacterial membrane structures (Cowan et al., 1999 and Cowan et al., 2008). Recent findings revealed that tea tree oil damages the cell membrane structure of E. coli, S.  and C. albicans.

Adiantum capillus

Aqueous extract of Adiantum capillus against Staphylococcus aureus exhibited maximum zone of inhibition 18±0.41 at 100 mg/ml and minimum zone of inhibition (6±0.03) was recorded at 25 mg/ml which significantly increased at 50 and 75 mg/ml where zone of inhibition was 9±0.31 and 14.0±0.14, respectively. Furthermore, against E. coli maximum zone of inhibition 12±0.21 was shown 100 mg/ml and minimum zone of inhibition (4±0.39) was recorded at 25 mg/ml which significantly increased at 50 and 75 mg/ml where zone of inhibition was 6±0.07 and 8±0.09 respectively. Against streptococcus agalactia exhibited maximum zone of inhibition 13±0.31 at 100 mg/ml and minimum zone of inhibition (5±0.32) was recorded at 25 mg/ml which significantly increased at 50 and 75 mg/ml where zone of inhibition was 7.0±0.43 and 11±0.34 respectively, while as, against K. pneumoniae maximum zone of inhibition 15±0.31was recorded at 100 mg/ml while as minimum zone of inhibition (5±0.12) was recorded at 25 mg/ml which significantly increased at 50 and 75 mg/ml where zone of inhibition was 7.0±0.21 and 9.0±0.14 respectively. Aqueous extract of Adiantum capillus exhibited high antibacterial activity against Staphylococcus aureus, E. coli followed by K. pneumonia and. and the findings corraborates with Hussain et al. (2014), Meenakshi et al. (2008), Ishaq et al. (2014) and Ansari and Ekhlasi-Kazaj (2012).

Nepata cataria

Aqueous extract of Napeta cataria against Staphylococcus aureus exhibited maximum zone of inhibition 15±0.33 at 100 mg/ml and minimum zone of inhibition (8.03±0.02) was recorded at 25 mg/ml which significantly increased at 50 and 75 mg/ml where zone of inhibition was 12.03±0.03 and 14±0.01 respectively, while as against E. coli exhibited maximum zone of inhibition 16.01±0.19 at 100 mg/ml and -(nil) zone of inhibition was recorded at 25 mg/ml which significantly increased at 50 and 75 mg/ml where zone of inhibition was 5.02±0.07 and 8.09±0.02 respectively. Against streptococcus agalactia exhibited maximum zone of inhibition 14.09±0.37 at 100 mg/ml and minimum zone of inhibition (4.90±0.02) was recorded at 25 mg/ml which significantly increased at 50 and 75 mg/ml where zone of inhibition was 7.03±0.04 and 10.01±0.04 respectively. Against K. pneumoniae maximum zone of inhibition 13.31±0.41 was recordedat 100 mg/ml and 3.01±0.03 zone of inhibition was recorded at 25 mg/ml which increased at 50 and 75 mg/ml where zone of inhibition was 5.03±0.12 and 9.03±0.1 respectively. Aqueous extract of Nepata cataria exhibited high antibacterial activity against Staphylococcus aureus, E. coli and the findings are in corroboration with the findings of Bandh et al. (2011). The antimicrobial activity of AENC may be related to the monoterpenoid component i.e. nepetalactone present in the extracts as confirmed by a study.

Levandula stoeches

Aqueous extract of Levandula stoeches against Staphylococcus aureus exhibited maximum zone of inhibition 18.0±0.33 at 100 mg/ml and minimum zone of inhibition (10.0±0.71) was recorded at 25 mg/ml which significantly increased at 50 and 75 mg/ml where zone of inhibition was 12.0±0.314 and 14±0.31 respectively, While as, against E. coli maximum zone of inhibition was recorded at 13.0±0.141 at 100 mg/ml and minimum zone of inhibition (6.0±0.14) was recorded at 25 mg/ml which significantly increased at 50 and 75 mg/ml where zone of inhibition was 8.0±0.414 and 8.0±0.414 respectively. Against streptococcus agalactia maximum zone of inhibition 14.0±0.191 was shown at 100 mg/ml and minimum zone of inhibition (8.0±0.32) was recorded at 25 mg/ml which increased at 50 and 75 mg/ml where zone of inhibition was 8.0±0.313 and 10.0±0.333 respectively. Against K. pneumoniae maximum zone of inhibition 11.0±0.181 at was recorded 100 mg/ml and 4.0±0.12 zone of inhibition was recorded at 25 mg/ml which significantly increased at 50 and 75 mg/ml where zone of inhibition was 8.0±0.419 and 9.0±0.00 respectively.

Borago officinalis

Based on the in vitro antibacterial sensitivity test of herbal aqueous extracts, three extracts, Fumaria indica, Adiantum capillus, Nepata cataria significantly inhibited bacterial growth under study and maximum inhibition was recorded at concentration 100 mg/ml, while the extracts, Levandula stoeches significantly inhibited bacterial growth under study however, due to increased cytotoxicity, it was ignored for further study. Moreover Borago officinalis has shown nil to non-significant bacterial growth inhibition activity in addition with cytotoxicity effect. Aqueous extract of Borago officinalis against Staphylococcus aureus exhibited maximum zone of inhibition 5.0±0.31 at 100 mg/ml and nil/no zone of inhibition  was  recorded  at  25  and  50  mg/ml  which  significantly  increased to 3.0±0.14 at 75 mg/ml while as, against E. coli maximum zone of inhibition 9.0±0.29 was exhibited at 100 mg/ml and nil/no zone of inhibition was recorded at 25 mg/ml which significanttly increased at 50 and 75 mg/ml where zone of inhibition was 4.0±0.31 and 6.0±0.11 respectively. Against streptococcus agalactia maximum zone of inhibition 1.0±0.13 at 100 mg/ml and nil/no zone of inhibition was recorded at 25, 50 and 75 mg/ml while as, against K. pneumoniae exhibited no/nil zone of inhibition at 25, 50, 75 and 100 mg/ml.

Conclusion:

The highest AST response was found in Fumaria indica followed by Adiantum capillus and Nepata cataria. Fumaria indica and Adiantum capillus can be used as a commercial herbal preperations as an alternative treatment for bacterial infections.

Table-1: In vitro antibacterial AST of aqueous herbal leaf extracts

Test extract Test organism Test concentration (zone of inhibition) (mg/ml) Standard drug                 (30 mg)
25 50 75 100
Fumaria indica A 9.0±0.51a 11.0±0.33a 17.0±0.19b 20.0±1.21c 23.11±0.42
B 11.0±0.22a 13.0±0.41a 19.0±0.16b 21.0±0.19b 28.00±0.59
C 5±0.32a 6.00±0.41b 12.0±0.04c 13.0±0.37c 19.00±0.21
D 4.0±0.90a 9.0±0.19b 12.0±0.57b 17.0±0.49c 18.00±0.21
Adiantum capillus A 6±0.03a 9±0.31a 14.0±0.14b 18±0.41c 23.11±0.42
B 4±0.39a 6±0.07a 8±0.09b 12±0.21b 28.00±0.59
C 5±0.32a 7±0.43a 11±0.34b 13±0.31b 19.00±0.21
D 5±0.12a 7±0.21a 9±0.14b 15±0.31c 19.00±0.29
Nepata cataria A 8.03±0.02a 12.03±0.03a 14±0.01b 15±0.33b 23.11±0.42
B 5.02±0.07a 8.09±0.02b 16.01±0.19b 28.00±0.59
C 4.90±0.02a 7.03±0.04b 10.01±0.04c 16.09±0.37d 19.00±0.21
D 3.01±0.03a 5.03±0.12a 9.03±0.1b 13.31±0.41c 19.00±0.29
Levandula stoeches A 10.0±0.71a 12.0±0.31a 14±0.31b 18.0±0.33c 22.0±0.12
B 6.0±0.14a 8.0±0.41a 8.0±0.414a 13.0±0.14b 17.0±0.43
C 8.0±0.32a 8.0±0.31a 10.0±0.33a 14.0±0.19b 13.0±0.77
D 4.0±0.12a 8.0±0.41a 9.0±0.00a 11.0±0.18a 20.0±0.37
Borago officinalis A 3.0±0.14a 5.0±0.31a 25.0±0.18
B 4.0±0.31a 6.0±0.11a 9.0±0.29b 16.9±0.33
C 1.0±0.13a 12.5±0.67
D 22.0±0.47

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

A= Staph. , B= E. coli, C= streptococci agalactia, D= K.pneumoniae

Acknowledgement:

I owe my thanks to SKUAST-K, Faculty of Veterinary Sciences and Animal Husbandry for providing me the necessary facilities to conduct the research program.

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