Antituberculor activity of Curcuminoids isolated from Curcuma longa Rhizomes

 

Rahul Shivarkar1,2*, Satish Bhise3, V Rama Mohan Gupta4

1Sinhgad Institute of Pharmaceutical Sciences, Lonavala - 410401 Maharashtra State, India.

2Jawaharlal Nehru Technological University, Hyderabad.

3Late Narayandas Bhawandas Chhabada College of Pharmacy, Satara - 415020 Maharashtra, State, India.

4Pulla Reddy Institute of Pharmacy Annaram (V) Gummadidala (M) Hyderabad - 502313 A.P.

*Corresponding Author E-mail: rahulshivarkar19@gmail.com

 

ABSTRACT:

Medicinal plants and their isolated Phyto-constituents offer a hope for developing alternate medicines for the treatment of microbial diseases especially tuberculosis. The present research work aimed to isolate and purify curcuminoids from Curcuma longa rhizomes extract and evaluated for anti-tuberculosis studies. The Curcuma longa rhizomes were collected, authenticated and processed for isolation, separation and purification of curcumminoids by using TLC, column chromatography, UV and analyzed by HPLC. The purified curcuminoids, evaluated for Anti-tubercular activity by Microplate Alamar Blue assay (MABA) and Lowenstein Jensen (L-J) medium. Studies on evaluation of anti-tubercular activity including multi drug resistant tuberculosis (MDR) were performed by using standard compounds such as Rifampicin, Ethambutol and Streptomycin. Preliminary phytochemical and pharmacognostical screening of Curcuma longa rhizomes showed the resence of various secondary metabolites. Various solvent at different polarity were pre-tested in TLC for separation of curcuminoids, chloroform: ethanol at 95:5 showed better resolution of Rf value at 0.75, 0.55, 0.27, as Curcumin (C), Demethoxycurcumin (DMC), Bisdemethoxycurcumin (BDMC) respectively. The purified curcuminoids fractions were obtained by silica gel column chirography and quantity by using UV. The whole extracts and isolated compounds (fractions), individually, exhibited, anti-tuberculosis activity, the proportion of inhibition of these phyto-constituents for M. tuberculosis strain, inhibition was found to be at concentrations such as 12.5µg/ml, 25µg/ml, 50µg/ml, etc. The extracts and fractions of curcuminoids individually showed significant anti-tubercular activity. The curcuminoids showed greater activity as compared to whole plant extracts.

 

KEYWORDS: Antitubercular activity, Curcuma longa, curcuminoids, Multi Drug resistance.

 

 


 

INTRODUCTION:

Tuberculosis (TB) has existed for millennia and remains a major cause of health problem which is one of the top 10 causes of deathworldwide. An estimated 10.0million (range, 9.0–11.1million) people fell ill with TB in 2018, a number that has been relatively stable in recent years (Global tuberculosis report 2019 WHO)1. Mycobacterium tuberculosis that are resistant to at least rifampicin and isoniazid, two key drugs in the treatment of the disease TB is Known as Multidrug Resistance Tuberculosis (MDR)1. Since 1980s, the number of TB cases throughout the world has been increasing rapidly due to the emergence of multi-drug resistant Mycobacterium tuberculosis (MDR-TB)2. In the case of acquired drug resistance only second-line drugs e.g. Capreomycin as Cycloserine, Kanamycin, Ethionamide, have been used which have significant side effects with approximately 50% cure rate. However, fluro-quinolones such as Ofloxacin, Norfloxacin can be used which are safer than above-mentioned second-line drugs but are equally costlier3. There have been only two new anti-TB drugs introduced (Bedaquiline and Delamanid have been discovered recently). Since 2015, there are many new cases of multidrug-resistant TB (MDR-TB) and continuous addition in patients with Rifampicin-resistant TB (RR-TB) who were also newly eligible for MDR-TB treatment4. In the case of acquired drug resistance only second-line drugs have to be used but are found equally costlier. There is an urgent need to search for and develop new, effective and affordable anti-TB drugs5. The World Health Organization (WHO) has defined traditional medicine (including herbal drugs) as comprising therapeutic practices that have been in existence, often for hundreds of years, before the development and spread of modern medicine and are still in use today6. Herbs have long held an important place in the treatment of ailments. Over 350 natural products, mainly plant species; have been used in traditional medicine and antimicrobial activities7. The active principles have provided leads in the development of several life saving drugs, which are in clinical use today. Medicinal plants and their isolated active principles or phyto-constituents offer a hope for developing alternate medicines for the treatment of microbial diseases especially tuberculosis. In present research work, an attempt is to isolate the active compounds from medicinally important plants extracts and evaluate them and whole extracts for their pharmacognostical study and antimicrobial/anti-tubercular activity.

 

EXPERIMENTAL:

Collection, Authentication of Selected Plants:

The selected plant rhizomes of Curcuma. longa (C. longa) were collected from forest of Ghats region of Maharashtra state (village Apti Tal. Maval) and authenticated by Gov. of India Ministry of Environment, Forest and Climate Change, Botanical Survey of India Pune and deposited in the herbarium (RSS-01/02/03/04). The rhizomes were harvested, washed thoroughly to remove impurities, cleaned and shade dried, and then ground to coarse powder

 

Pharmacognostical study of rhizomes of Curcuma. Longa:

Macroscopy:

The dried rhizomes evaluated for macroscopic or morphological, organoleptic characters like appearance, colour, odour taste and other external characteristics

Microscopy:

The C. longa evaluated for microscopical studies with respective study of their Transverse section were cut by free hand sectioning and numerous sections examined microscopically.

 

Photographs of the microscopical sections were captured with the help of Olympus tri-ocular research microscope CX- 21I with Dig eye camera using Caliper plus version 4. 2 software.

 

Physical evaluation:

The C. longa rhizomes are were further evaluated for following parameters

1.     Total Ash value

2.     Acid Soluble ash value

3.     Water soluble ash value

4.     Alcohol soluble extractive value

5.     Water soluble extractive value

6.     Loss on drying

 

Extraction of plants:

Method:

Soxhlet Extraction.

 

Solvents used:

The HPLC graded solvents such Hexane, Chloroform, Ethyl acetate, Methanol, and Acetone Aqueous are used for extraction. Ethanolic extracts prepared and stored at -20˚C. Extracts dissolved in 1% DMSO (wt/vol) for anti-tubercular activity.

 

Phyto-chemical Investigation:

The plant extracts are investigated for the presence of following secondary metabolites Carbohydrates, Glycosides, Alkaloids, Tannins, Resins, Terpenoids Volatile oil and Steroids

 

Estimation of curcuminoids:

Preparation of Sample:

Weighed accurately 25mg sample and dissolved in 25ml ethanol. From this pipette out 1ml and diluted to 5ml with ethanol.

 

Separation of curcuminoids by TLC using different solvent system:

Ethanolic extract evaluated by TLC for presence of three curcuminoids. The TLC pre-coated silica gel (Merk-60 F254, 0.25mm thick) plate were developed using a Camag twin- trough glass tank which was pre-saturated with the mobile phase for 1 hour and each plate was developed to a height of about 10cm. The composition of mobile phase was optimized by using different mobile solvents of varying polarity. After development plates were removed and dried and spots were visualized in UV light.

 

Column chromatography:

Sample preparation:

100gm of fine powdered rhizome were subjected to soxhlet extraction and solvent used were Ethanol for 6 hours. The extract was filtered and concentrated in rotary evaporator, yielded olerosin was precipitated with petroleum ether and vaccum dried, this crude curcuminoid mixture contained curcumin, demethoxycurcumin, bisdemethoxycurcumin.

 

Silica gel column chromatography:

Ethanolic extract was subjected to column chromatography in silica gel (60-120 mesh) glass column. About 5gm of crude Curcuminoids were mixed with 8gm of silica gel and loaded onto the column of 46×2cm and eluted with chloroform followed by chloroform :methanol with increasing polarity. All the collected fractions were subjected to TLC silica gel 60 F254 plate using chloroform :methanol (95:5) as the developing solvent system and detected as yellow spots. And similar fractions with the Rf values were pooled and the organic solvent was removed by rotary evaporator. The total curcuminoid content of each curcuminoid collected were analysed by UV spectrophotometry at 420nm.

 

Anti-tubercular Activity of plant extracts and isolated compounds:

1.     Assessed against M. tuberculosis using Microplate Alamar Blue assay (MABA).:

MABA is non-toxic and uses a thermally stable reagent and shows good correlation with proportional and BACTEC radiometric method. Briefly, 200μl of sterile lavonoid water was added to all outer perimeter wells of sterile 96 wells plate to minimized evaporation of medium in the test wells during incubation. The 96 wells plate received 100μl of the Middle brook 7H9 broth and serial dilution of compounds was made directly on plate. The final drug concentrations extract and all isolated curcuminoids tested were 50 to 0.8μg/ml. Plates were covered and sealed with para film and incubated at 37şC for five days. After this time, 25μl of freshly prepared 1:1 mixture of Almar Blue reagent and 10% tween 80 was added to the plate and incubated for 24 hrs. A blue color in the well was interpreted as no bacterial growth, and pink color was scored as growth.

 

The MIC was defined as lowest drug concentration which prevented the color change from blue to pink.

 

Standard Strain used: Mycobacteria tuberculosis (Vaccine strain, H37 RV strain): Standard values for the Anti-Tb test which was performed by using Pyrazinamide- 3.125μg/ml Isoniazide-3.125μg/ml Streptomycin- 6.25μg/ml as standard Drugs.

 

2.       Anti-tubercular activity of plant extracts and isolated compounds against Mutidrug registrant MTB isolates using Lowenstein Jensen (L-J) medium

·         Preparation of extracts and curcuminoids samples: The fresh water extract of Curcuma longa rhizome is prepare by crushing the rhizomes in mortor and pestle by using sterile distilled water in ration 1: 1 and is prepared 12.5µg/ml, 25µg/ml, 50µg/ml, etc

 

·         Mycobacterial strains/isolates:

Reference drug, susceptible strain M. Tuberculosis H37Rv as control, multi- drug résistant isolates (DKU-156 and JAL 1236) and and fast growing mycobacterial pathogen M. fortuitum (TMC-1529)

 

·         Assay protocol:

Antimicrobial assays were performed in Lowenstein Jensen (L-J) medium and determination of Colony forming units (cfu)

 

Antimicrobial assays were performed in Lowenstein Jensen (L-J) medium .Determination of Colony forming units (cfu) on Lowenstein-Jensen (L-J)-The ten-fold dilution of standard 1 mg/ml M. tuberculosis suspension were streaked on L-J medium for determining cfu in the presence and absence of plant extracts. An M. tuberculosis suspension of 1 mg/ml is equivalent to MacFarland standard-120. One loopful (6 µl) of this suspension was streaked on the L-J slants using 3 mm external diameter loop. Reagents of L-J media included potassium di hydrogen phosphate anhydrous, magnesium sulphate anhydrous, magesium citrate, L-asparagine, glycer, and malachite green.

 

The plant extract and isolated curcuminoids sample solutions incorporated in the medium at concentration of 2 per cent v/v and 4 per cent v/v (2 ml and 4 ml of fresh plant extract was dissolved into 100 ml of culture medium) prior to inspissations. The medium set inoculated with the standard bacterial suspension and incubated at 37°C for 42 days. Reading was taken weekly. For comparison, extract free control slants were used. Susceptibility testing of MDR isolates was also performed against standard drugs like: rifampicin and isoniazid in the same batch of media for comparison of cfu on drug free controls. Each test was done in duplicate. Percentage inhibition was calculated by mean reduction in number of colonies on extract containing as compared to extract free controls. All the experiments (including those in which activity against MDR and sensitive strains were studied) were carried out in triplicate and repeated on three separate occasions. The experiments were performed in a Bio safety level three (BSL-3) laboratory facility available in Biocyte Institute of Research and Development, Sangali (Maharashtra)

 

RESULT AND DISCUSSION:

Pharmacognostic study of part of plants:

The authenticated plants (drugs) are evaluated for their morphological, microscopical study and physico-chemical properties. The evaluated drugs showed identical, morphological, microscopical characteristics and physic-chemical properties.

 

Table: 1 Physical evaluations of Part plants

Parameters of evaluation

(Values in percentages)

Curcuma longa rhizomes

Total Ash value

13.50

50 v

Acid insoluble ash value

1.30

Water soluble ash value

4.30

Alcohol soluble extractive value

18.25

Water soluble extractive value

21.9

Loss on drying

4.10

 

Phyto-chemical Investigation:

The morphologically, microscopically and physio-chemicaly evaluated plants are further processed for extraction. The extracts of individual plant extracts phytochemicaly evaluated for the presence of phyto-constituents

Table 2: Phyto-chemical Investigation

Phytochemical constituents

C. longa

Rizhome Extact

 

Alkaloidis

-

 

Carbohydrate

-

 

Glycosides

+

 

Flavonoids

-

 

Tannins

-

 

Proteins

+

Amino acids

-

Fats and fixed oils

-

Sterols

-

Terpenoids

+

Volatile oil

+

 

Separation and purification of fraction of C. longa extract by TLC and Column chromatography using different solvent system:

 

Table 3: Mobile phases for separation different fraction C. longa extract by TLC of and their RF values (fraction 1, 2 and 3)

TLC mobile Phase

Ratio

Rf values

1

2

3

Benzene: ethylacetate

18 : 2

0.79

0.69

0.61

Dichloromethane: methanol

19 : 1

0.8

0.7

0.6

Chloroform: methanol

19 : 1

0.75

0.55

0.27

Each plate was developed to a height of about 8cm.


 

Purification of each curcuminoids by column chromatography:

Table 4: Silica gel column chromatography elution profile

Fractions numbers

Total volume collected (Ml)

Curcuminoids present

Weight of extract (mg)

Total Curcuminoid by UV spectroscopy

1 to 31

240

C

906.4

84%

32 to 40

360

C+DMC

173.5

22%

41 to 67

080

DMC

597.5

86%

68 to 75

320

DMC+BDMC

192.7

46.6%

76 to 95

800

DMC+BDMC

390.5

80.61%

 


Different compositions of mobile phase were tested in TLC for the separation of individual curcuminoids and its Rf values were determined shown in table 3. The desired resolution of separation was achieved using chloroform: methanol 95:5 as the mobile phase. The Rf value of curcuminoids were 0.75, 0.55, and 0.27, for C, DMC, BDMC respectively. Better resolution of Rf value showed that chloroform and methanol can be suitable solvent for the separation of compounds in column chromatography. In the present study acetone extract was precipitated with petroleum ether and yielded crude curcuminoids were subjected to column chromatography the elution was done using chloroform followed by chloroform: methanol with increasing polarity and the fractions obtained were tested with TLC. Fractions showed same pattern in TLC were pooled and concentrated. The composition of the fractions collected during column chromatographic separation of crude curcuminoids and the concentrated fractions were tested for determination of total curcuminoids by UV spectroscopy is shown in Table 4.


 

Anti-tubercular Activity of plant extract and isolated compounds: Microplate Alamar Blue assay (MABA).

Table 5: Anti-tubercular Activity of plant extract and isolated compounds: Microplate Alamar Blue assay (MABA). (S: Sensitive R: Resistant).

S. No.

Sample

50 µg/ml

25 µg/ml

12.5 µg/ml

6.25 µg/ml

3.12 µg/ml

1.6 µg/ml

0.8 µg/ml

1

C.Longa extract

S

R

R

R

R

R

R

2

Curcumin C

(sample1)

S

S

S

S

R

R

R

3

DMC

(Sample 2)

S

S

R

R

R

R

R

4

BDMC

(Sample 3)

S

R

R

R

R

R

R

 

Fig.1: Anti-tubercular Activity of isolated compounds: Microplate Alamar Blue assay (MABA). A blue color in the well was interpreted as no bacterial growth, and pink color was scored.

 

Anti-tubercular activity of plant extracts and isolated compounds against Mutidrug registrant MTB isolates using Lowenstein Jensen (L-J) medium:

Table 6: Anti-tubercular activity of plant extract and isolated curcumioids sample

L-J proportion method

Plant name

Extract

M.TB Strain

Mean cfu on

media % Inhibition

Control

Plant Extract

Plant Extract

2% v/v

4% v/v

2% v/v

4% v/v

C. Longa

Water

H37Rv

DKU-156

JAL 1236

M. fortuitum

52

22

72

2

27

6

25

3

15

2

23

0

60

65

64

0

72

70

68

0

Curcumin (Sample 1)

Water

H37Rv

DKU-156

JAL 1236

M.fortuitum

65

25

75

10

35

5

28

7

20

2

25

5

68

65

55

2

82

70

65

2

DMC (Sample 2)

Water

H37RV

DKU-156

JAL 1236

M.fortuitum

66

25

72

4

30

6

22

1

14

5

24

64

60

64

78

75

66

BDMC (Sample3)

Water

H37RV

DKU-156

JAL 1236

M. fortuitum

68

25

75

3

34

4

20

0

10

2

24

0

80

75

65

0

90

85

66

0

 


The results showed inhibitory effect on all the strains of Mycobacterium tuberculosis used in this study. The aqueous sample of isolated curcuminoids were effective as compared to whole extracts.

 

DISCUSSION:

In the study the selected plant were collected, authenticated and processed for extraction. The extracts of plants were further processed for Phyto-chemical investigation, isolation, separation and identification, purification and characterization of Phyto-constituents. The extracts and phyto-constituents were screened for their antimicrobial including anti-tubercular activity. The plant extracts and isolated curcuminoids samples exhibited promising anti-tubercular activity. The isolated Phyto-constituents showed greater antimicrobial/anti-tubercular activity as compare to whole plant extracts. The study showed possibility of conducting studies on in-vivo antimicrobial/anti-tubercular activity of the isolated compounds.

 

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Received on 18.11.2020         Modified on 08.12.2020

Accepted on 21.12.2020  ©AandV Publications All right reserved

Res. J. Pharmacognosy and Phytochem. 2021; 13(1):5-10.

DOI: 10.5958/0975-4385.2021.00002.9