Antimicrobial Activity of the protein fraction obtained in the extraction of Curcumin

 

T. Sharanya Nair, Meghana R, Shlini P*

Department of Chemistry (PG Biochemistry). Mount Carmel College, Autonomous. #58. Palace Road. Bangalore - 560052. Karnataka. India.

*Corresponding Author E-mail: shlinip1@gmail.com

 

ABSTRACT:

Curcumin is a phytopolyphenolic component and active principle of turmeric. Curcumin inhibits the formation of reactive oxygen species formation and is anti-inflammatory. It inhibits the various enzymes involved in inflammation. Curcumin is also known to be an antimicrobial, antioxidant, anti-inflammatory to inhibition of angiogenesis, chemopreventive and anticancer agent. However in spite of all its therapeutic properties in vitro, it fails to be an active drug in vivo due to its poor bioavailability, rapid metabolism and an overall poor pharmacodynamic and pharmacokinetic activities. Studies are uncertain whether these compounds act independently, additively or synergistically with curcumin to exhibit the above mentioned properties. Inflammation of either upper urinary tract or lower urinary tract caused by invasion of uropathogens is recognised as Urinary Tract Infection (UTI), of which infection of the lower urinary tract is the most common and is characterised by asymptomatic bacteraemia, pelvic pain, frequent urination, urge to urinate, etc. Women are more susceptible to UTI than men owing to the shorter length of urethra. Recurrent UTI is seen to be a characteristic of infection by MDR strains of uropathogens, developed due to chronic illness, prolonged and frequent hospitalisation, previous history of endoscopic surgery and diabetes mellitus type 2. The present study is aimed at studying the effect of protein extracts of curcumin from the plant sample yielding the maximum inhibition against the uropathogen isolated from urine sample obtained from a diabetic patient suffering from UTI. The active principle of turmeric, curcumin and curcumin free turmeric extract were tested in vitro for its antibacterial activities against the bacterial isolate and were found to be nil. However, the protein extracts of the crude curcumin showed a positive response in inhibiting the isolate.

 

KEYWORDS: UTI, MDR, Pseudomonas aeroginosa, Curcuma longa, curcumin, curcumin free turmeric, ampicillin.

 

 


INTRODUCTION:

Curcumin, a phytophenol is a natural component of the rhizome of turmeric (Curcuma longa) and is considered as the active principle component. Its biological effects range from antimicrobial, antioxidant, anti-inflammatory to inhibition of angiogenesis and is also shown to possess specific antitumoral1.

 

However, these effects are not expressed in vivo due to its poor pharmacodynamic and pharmacokinetic activities2. This lead to further investigation of other components of turmeric with potential medical activities leading to the discovery of compounds such as β sesquiphellandrene, elemene, turmerin, tumerone, furanodiene, curodiene, bisacurone, calebin A and germerone which may act independently or along with curcumin to produce the desired effects3.

 

Infection of part of the urinary tract is called urinary tract infection or UTI. Infection of lower urinary tract is the most common and is accompanied by asymptomatic bacteraemia, frequent urination, pelvic pain, etc. An upper urinary tract infection is an infection of the kidney4. Organisms causing UTI are called uropathogens and include both gram positive bacteria such as Staphylococcus sp, Streptococcus sp and Enterococcus faecalis and gram negative bacteria like E. coli, Klebsiella sp, Enterobacter sp, Proteus sp, Serratia sp and Pseudomonas sp. Most of the uropathogens have the potential of gaining resistance to drugs and thus pose a risk of recurrence of the infection also indicating that it is a nosocomial infection placing at risk patients who have had prolonged illness, frequent hospitalisation and Type 2 diabetes mellitus5 .

 

Pseudomonas aeruginosa is a gram negative pathogen affecting both flora and fauna equally. P. aeruginosa is a multidrug resistant pathogen which is made possible by mechanisms such as restricted uptake and influx; drug inactivation and changes in drug targets. This makes it to be of considerable medical importance and is found to be associated with serious illnesses hospital-acquired infections such as ventilator-associated pneumonia and various sepsis syndromes6. P.aeruginosa has a large genome of 6.26 Mbp, encoding 5567 genes which confers its high adaptability to antibiotics used.

 

The present study is aimed at studying the effect of protein extracts of curcumin from the plant sample yielding the maximum inhibition against the uropathogen isolated from urine sample obtained from a diabetic patient suffering from UTI.

 

MATERIALS AND METHOD:

Plant sources:

Rizomes of Curcuma longa, Curcuma aromatica, Curcuma zedoarua and Zingiberaceae officionale and Crocus sativus were purchased from various supermarkets at Bengaluru, Karnataka. India.

 

Extraction of proteins from curcumin:

A 20% aqueous extract was prepared by homogenizing Curcuma longa and incubated for 30 minutes. This was then filtered and the filtrate was centrifuged at 10000 rpm for 20 minutes.

 

Ammonium sulphate precipitation:

Ammonium sulfate fractionation was carried out by adding the small amount of powdered ammonium sulfate to the supernatant obtained by constant stirring over magnetic stirrer at 4C to obtain 0 - 30% saturation. The solution allowed to stand for 30 mins at 4C followed by centrifugation at 10,000 rpm for 20 mins. The pellet obtained was dissolved in 5 ml of phosphate buffer (pH 7 [10mM]). The supernatant was collected and subjected to 30 - 60 % and 60 - 80% ammonium sulphate fractionation as described above, followed by centrifugation at 10,000 rpm for 20 mins. The pellet was dissolved in 5 ml of phosphate buffer (pH 7 [10mM]). The fractions (0 - 30%, 30 - 60 % and 60 - 80%) were dialyzed against 10 mM phosphate buffer for 30 - 60 % for 8 hours, with two changes in the buffer. The dialysate was centrifuged at 10,000 rpm for 20 mins to remove the insoluble residue and the supernatant was used for determination of protein content.

 

Estimation of protein:

The 30%, 60% and 80% pellets obtained were used to determine the protein content by the method of Lowry et al 1951 with bovine serum albumin as standard7.

 

Bacterial source:

Urine sample was collected under aseptic conditions from a diabetic patient suffering from UTI and was being treated at Bhagwan Mahaveer Jain Hospital, Bengaluru. Samples were collected in sterile and clean urinary containers and cultivated for identification.

 

Colony Development of urine samples:

The sample were inoculated by streaking on MacConkey agar and incubated at 37°C for 48 hours and were observed for right pink coloured colonies. Four different well isolated distinct colonies were observed. Pure cultures of these colonies were obtained by sub culturing on MacConkey by quadrant streaking and slant streaking. Each isolate was given a number before its identification and these numbers were maintained throughout.

 

Biochemical Identification of isolates:

Sub cultures of isolates were further identified and characterized by various biochemical tests namely indole, MR-VP, citrate utilization, sugar fermentation test8, urease test, catalase test and oxidase test. The morphology was identified using Gram’s staining method by Hans Christian Gram9.

 

Antimicrobial test:

Antimicrobial sensitivity of the UTI subcultures was tested by well diffusion method. Each of the four subcultures were inoculated in a nutrient broth and was incubated overnight. 0.1 ml of each of the overnight grown cultures were spread onto nutrient agar plates and four wells were punched and 35μl of positive control (ampicillin), negative control (DMSO), pellet 1 (30% protein pellet), pellet 2 (60% protein pellet) and pellet 3 (80% protein pellet) were loaded and incubated overnight at 37˚C.

 

RESULT AND DISCUSSION:

Urinary tract infection is an infection of part of the urinary tract caused by pathogenic invasion resulting in an inflammation of the urothelium. Proliferation of these invading pathogenic bacteria in the urinary tract cause UTI. The extent of the inflammation depends upon the part of the urinary tract infected, etiologic organisms, severity of infection and the state of the patient’s immune system. An upper UTI affects the kidney while the lower UTI affects the urinary tract and is identified by asymptomatic bacteruria, pelvic pain, frequent urination, inconsistency, scanty urination and rectal pain in men. UTI generally affects people of all gender and age. Owing to the poor pharmacodynamic and pharmacokinetic activities of curcumin and its poor therapeutic properties in vivo researches have tried to tap the value of compound apart from curcumin. Studies indicate curcumin free extract to exhibit antidiabetic, anticancer and anti inflammatory activities. These compounds could be exhibiting their properties uniquely, additively, or synergistically with curcumin.

 

Estimation of proteins by Lowry’s method:

Protein estimation of the protein extracts from curcumin indicated that the 30% extract (pellet 1) to have the highest amount of protein of 480µg when compared to 60% and 80% ammonium sulphate precipitation. Thus on protein estimation, 50ml of the extract was found to contain 24mg of protein equivalent to bovine serum albumin, which corresponds to the amount of protein present in 10g of Curcuma longa (Fig. 1).

 

Colony Characteristics:

MacConkey media is a differential and selective culture media which is used for the growth of Gram negative and Enteric bacteria. Differentiation between the bacteria is based on lactose fermentation. Quadrant streaking of a loopful of the urine sample was carried out on MacConkey agar plate and was incubated at 37˚C for 48 hours. At the end of the incubation period, four morphologically different colonies were identified and their characteristics were observed and were tabulated as in table 1.

 

Fig 1: Protein estimation of the extract (30%, 60% and 80%) by Lowry method.

 

Biochemical tests:

Each of the obtained colonies were then subcultured into four different MacConkey agar plates and was maintained for further use (Fig 2). On Gram staining all the four colonies were observed to be Gram positive rods. The four subcultures were then subjected to an array of biochemical tests and the results obtained are as tabulated in table 2.

 

Fig 2: Quadrant steak of urine sample on MacConkey agar.

 

 


Table 1: Colony Characteristics

Col No.

Size

Colour

Shape

Margin

Elevation

Transparency

Texture

Gram character

1.

0.3cm

Pale yellow

Round

Entire

Flat

Opaque

Butyroid

Negative bacilli

2.

0.3cm

Pink

Scalloped

Scalloped

Flat

Opaque

Butyroid

Negatve bacilli

3.

0.5cm

Yellow

Round

Crenate

Flat

Translucent

Butyroid

Negative bacilli

4.

0.2cm

Pale yellow

Round

Entire

Flat

Translucent

Butyroid

Negative bacilli

 


Table 2: Biochemical tests

 

Starch hydrolysis

Casein hydrolysis

Indole

MR

VP

Citrate utilization

Catalase

Oxidase

Urease

Colony 1

-

+

-

-

+

+

+

+

+

Colony 2

-

+

-

-

+

+

+

+

+

Colony 3

-

+

-

-

+

+

+

+

+

Colony 4

-

+

-

-

+

+

+

+

+

 


Antimicrobial test:

Antimicrobial sensitivity test was carried out on four subcultures with E.coli as the control against DMSO (negative control), ampicillin (positive control), curcumin extract, curcumin free turmeric extract, filtrate and pellet by agar well diffusion method which indicated that the four subcultures isolated from the UTI sample were not susceptible to any of the chemicals loaded into the well. However, E.coli was found to give a 1.6cm zone of inhibition with ampicillin and the results were tabulated (Table 3).

 

Antimicrobial sensitivity test was carried out on four subcultures with E.coli as the control against DMSO (negative control), ampicillin (positive control), pellet 1 (30%), pellet 2 (40%) and pellet 3 (80%) by agar well diffusion method (Table 4). After an overnight incubation at 37˚C, the plates were observed for the presence of inhibition zones. The plate inoculated with E.coli was found to have been inhibited by ampicillin and the 30% protein precipitate extract of curcumin by about 1.8 and 0.6cm respectively. The other four subculture plates were observed to have no zone of inhibition by ampicillin except colony 4 which showed a 0.5cm zone of inhibition. However, a better zone of inhibition was observed by the 30% protein precipitate extract of curcumin in all the four UTI isolates by 0.8cm, 0.6cm, 0.6cm, 0.6cm and 0.6cm respectively.


 

Table 3: Antimicrobial susceptibility by agar well diffusion method.

Culture

Negative control cm

Positive control cm

Curcumin extract cm

CFT cm

Filtrate cm

Pellet cm

E.coli

NIL

1.6

NIL

NIL

NIL

NIL

Colony 1

NIL

NIL

NIL

NIL

NIL

NIL

Colony 2

NIL

NIL

NIL

NIL

NIL

NIL

Colony 3

NIL

NIL

NIL

NIL

NIL

NIL

Colony 4

NIL

NIL

NIL

NIL

NIL

NIL

 

Table 4: Antimicrobial susceptibility test using protein fractions.

Culture

Negative control cm

Positive control cm

Pellet1 cm

Pellet2 cm

Pellet3 cm

E. coli

NIL

1.8

0.6

NIL

NIL

Colony 1

NIL

0.4

0.8

NIL

NIL

Colony 2

NIL

NIL

0.6

NIL

NIL

Colony 3

NIL

NIL

0.6

NIL

NIL

Colony 4

NIL

0.5

0.6

NIL

NIL

 


The results obtained indicated that extraction of curcumin from the organic solvent methanol is found to be more efficient due to its polyphenolic nature and the extracts from C.longa indicated highest value of curcumin among the members of the tested Zingiberaceae family. The collected urine sample on culturing and analysis identified the isolates to be Pseudomonas aeruginosa which indicated the chronic illness and a direct relation to the length of hospitalisation and the extent of advancement of the diabetic condition of the patient and acquisition of MDR strains of the organism most probably acquired by the patient as a nosocomial infection as are the risk factors of Pseudomonas aeruginosa10. The estimation of in vitro antimicrobial activity of crude curcumin extract was observed to be nil which was contradictory to the results obtained from a study much inclined to the review Nelson et al.,11. The in vitro testing of antimicrobial properties of CFT extract also showed no positive results which indicated that though CFT was observed to have anticancer and anti-inflammatory activities Aggarwal et al., showed no antimicrobial activities12. Further probe into the antimicrobial properties of protein extracts of curcumin showed appreciable results by the 30 % protein extract at comparatively higher levels than ampicillin on the isolated MDR uropathogens. This indicates that Pseudomonas aeruginosa has not developed resistance mechanisms against the natural product.

 

ACKNOWLEDGEMENT:

The authors wish to acknowledge Department of Chemistry (PG Biochemistry) and the Management of Mount Carmel College Autonomous, Bengaluru for funding this project and offering their facilities for the analysis.

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Received on 27.05.2019                    Modified on 20.06.2019

Accepted on 09.07.2019                   ©AJRC All right reserved

Asian J. Research Chem. 2019; 12(4):199-202.

DOI: 10.5958/0974-4150.2019.00037.3 9