INTRODUCTION:

Inflammation is considered as a complex host reaction to foreign substances or cell and tissue injury. If such infection is not subside, eliminated or the tissue damage remains, then the inflammation can become chronic that can cause many infections. Inflammation can be caused by a various physical reactions that may be triggered by the immune system in response to either physical injury or an infection^1,2. Acute inflammation lasts for shorter duration often causes noticeable symptoms, such as pain, redness, or swelling³.

Where as chronic inflammatory symptoms are usually subtler⁴. Some examples of pathogens that include bacteria, fungi, viruses etc they can grow and multiply quickly. Infectious arthritis is one of such pathogenic condition in which specific bacteria also can spread to joints in turn causes skin and soft infections⁵. S. aureus is a gram-positive round shaped bacterium belongs to the member of Firmicutes causes a multitude of infections include Lung infections, Skin infections, Bone infections such as osteomyelitis, Blood infections etc⁶. Pathogenic strains often promote infections by producing virulence factors such as potent protein toxins, and the expression of a cell-surface protein that binds and inactivates antibodies. The emergence of antibiotic-resistant strains of S. aureus such as methicillin-resistant S. aureus (MRSA) is a worldwide problem in clinical medicine. S. aureus is the bacterium commonly responsible for all major bone and joint infections^7-8. This manifests in one of three forms: osteomyelitis, septic arthritis, and infection from a replacement joint surgery^9-10. In other way, Escherichia coli (E. coli ) is a Gram-negative, anaerobic, rod-shaped, bacterium of the genus Escherichia that is generally found in the lower al part of GIT of warm-blooded organisms¹¹. Recent research revealed that, virulent forms of E. coli can cause many ailments such as gastroenteritis, urinary tract infections (UTI), neonatal meningitis, hemorrhagic colitis, and Crohn's disease etc. Boswellia serrata belongs to family Burseraceae a native plant of India. This plant posse a broad range of Pharmacological properties and has been used in many traditional systems of medicine^12-13. The major part of this plant possess therapeutic effect is gum, that consists of volatile oil, water soluble gum (polysaccharides), lipophilic terpenes and insoluble matter. The polysaccharide is abundant in neutral sugars and composed of galactose, arabinose, xylose and d-glucuronic acid etc. The toxicity and safety studies have established that the gum is non-toxic and safe for use in different animals^14-15. Based on the scientific evidence, the present study has been undertaken to evaluate the anti bacterial and In vitro anti inflammatory potential of Boswellia serrata standardized extract which will pave the way for the future research as well as for development of newer delivery system.

MATERIALS AND METHODS:

2.1. Materials:

All the chemicals used in this study were of analytical grade. Dimethyl Sulfoxide (DMSO), Hyaluronidase, hyaluronic acid was procured from Sigma – Aldrich, Bangalore. Peptone, Yeast extract, Sodium chloride were obtained commercial sources (from these chemicals were used in experiment without any further purification. Boswellia seratta standardized extract was a kind gift sample from Sunpure, New Delhi, India.

Methodology:

Anti-Bacterial Analysis:^16-17

The anti-bacterial potential of Boswellia seratta standardized extract was carried out by Threlfall E.J.et al and Walker R.D.et al. briefly, The media was prepared by using: Peptone-10 g, NaCl-10g and Yeast extract 5g, Agar 20g in 1000 ml of distilled water. The stock cultures of bacteria were revived by inoculating in broth media and grown at 37ºC for 18 hrs. The agar plates of the above media were prepared and wells were made in the plate. Each plate was inoculated with 18 h old cultures (100 μl, 10-4 cfu) and spread evenly on the plate. After 20 min, the wells were filled with of compound at different volumes. All the plates were incubated at 37ºC for 24 h and the diameter of inhibition zone were noted.

Evaluation of Anti-inflammatory Potential:¹⁸

The anti-inflammatory potential of Boswellia seratta standardized extract was carried out according to the method described by Ling S.K et al. briefly, The assay medium consisting of 3 - 5U hyaluronidase in 100μl of 20mM sodium phosphate buffer (pH 7.0) with 77mM sodium chloride, 0.01% BSA was pre incubated with different of the test compound for 15 min at 37°C. The assay was commenced by adding 100μl hyaluronic acid (from Sigma–Aldrich, Bangalore; 0.03% in 300mM sodium phosphate, pH 5.35) to the incubation mixture and incubated for a further 45 min at 37°C. The undigested hyaluronic acid was precipitated with 1ml acid albumin solution made up of 0.1% bovine serum albumin in 24mM sodium acetate and 79mM acetic acid (pH 3.75). After standing at room temperature for 10 min, the absorbance of the reaction mixture was measured at 600 nm. The absorbance in the absence of enzyme was used as the reference value for maximum inhibition. The inhibitory activity of test compound was calculated as the percentage ratio of the absorbance in the presence of test compound vs. positive control. The enzyme activity was checked by control experiment run simultaneously, in which the enzyme was pre incubated with 5μl vehicle instead (DMSO), and followed by the assay procedures described above. Compound was tested in a range of 100μg -1000μg in the reaction mixture. Indomethacin (Indo) was used as reference standard

RESULTS:

The results are presented in following table as diameter of inhibition zones in mm.

Table.1:

Organism	25	50	100	250	500	1000	MIC in μg
S.aureus	0	0	0	0	3	5	1000

Table.2: Anti bacterial effect of Boswellia serrata extract using E.coli

Organism	25	50	100	250	500	1000	MIC in μg
E.coli	0	0	0	4	2	3	1000

Table.3: Zone of inhibition calculated subtracting inhibition to Sample from Inhibition to Ethanol.

Organism

25 μg

μg

100μg

200

μg

400

μg

800

μg

MIC μg

E. coli

S. aureus

Table.4: Percentage inhibition of hyaluronidase enzyme by Boswellia serrata extract.

Percentage(%) hyaluronidase inhibition
Concentration	Sample	Indomethacin
10μl	7.59542	42.86
50μl	72.67176	87.95
100μl	84.84733	98.21

Fig.1: Graphical presentation of anti-inflammatory assay of Boswellia seratta

Plate.1: Zone of inhibition (Sample) against S.aureus

Plate.2: Zone of inhibition (Ethanol) against S.aureus

Plate.3: Zone of inhibition (Ciprofloxacin) against Methicillin resistant S.aureus

Plate.4: Zone of inhibition (Sample) against E.coli

Plate.5: Zone of inhibition (Ethanol) against E.coli

Plate.6: Zone of inhibition (Ciprofloxacin) against E.coli

DISCUSSION AND CONCLUSION:

The minimum inhibitory concentration (MIC) of the Boswellia serrata standardized extract and its various fractions were determined by the agar dilution method. Two strains of Staphylococcus aureus i.e. one standard strain and another Methicillin resistant S. aureus as well as standard strain of E.Coli was used in the present study to evaluate the anti bacterial potential. The results for anti bacterial activity of Boswellia serrata standardized extract against both the strains of S. aureus are presented in table no.1, plate no.1, plate no.2 and plate no.3 as diameter of inhibition zones in mm. The results for antibacterial activity revealed that the extract at 500 and 1000μg showed 3mm and 5mm of inhibition of zones respectively against S. aureus. Similarly, Anti bacterial effect of Boswellia serrata extract was evaluated against bacteria E.coli. The results were depicted in table no.2 and plate no.4, plate no.5 and plate no.6. It has been observed that, the extract at concentration of 250,500 and 1000μg showed 4mm, 2mm and 3mm of inhibition of zones respectively against E.coli. The Zone of inhibition was also calculated subtracting inhibition to Sample from Inhibition to Ethanol and values were tabulated in table no-3. The in-vitro anti inflammatory activity of Boswellia serrata extract was evaluated by hyaluronidase inhibition assay. The percentage inhibition of hyaluronidase enzyme by Boswellia serrata extract was mentioned in table no.4 and graphically represented in fig.no.2. The result revealed that, the extract at 100 μg showed 84.84% hyaluronidase inhibition in compared with 98.21% by standard drug indomethacin. Hence from the present study it has been concluded that the Boswellia serrata extract may provide a promising antimicrobial agent for therapeutic applications against S. aureus and E.coli. Further studies are required for investigation of the effect of the extract on cell lines to verify the bioactivity in animal models.

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Received on 16.03.2019 Modified on 09.05.2019

Research J. Pharm. and Tech 2020; 13(3): 1079-1082.

DOI: 10.5958/0974-360X.2020.00198.5