INTRODUCTION:

Tuberculosis (TB) is a contagious disease. Like the common cold, it spreads through the air. Only people who are sick with TB in their lungs are infectious. The World Health Organization (WHO) estimates that the largest number of new TB cases in 2005 occurred in the South-East Asia Region, which accounted for 34% of incident cases globally. However, the estimated incidence rate in sub-Saharan Africa is nearly twice that of the South-East Asia Region, at nearly 350 cases per 100 000 population. Currently, one third of the world’s population is infected with M. tuberculosis and each year there are 2 - 3 million deaths worldwide caused by the disease ⁽¹⁾. TB is a leading cause of death among people with human immunodeficiency virus (HIV). Individuals infected with HIV are very susceptible to TB and often develop this disease before other manifestations of AIDS become apparent ^(2;
3).

Today, strains of TB that are resistant to all major anti-TB drugs have emerged. The emergence of resistance to antimicrobials, though is a natural biological occurrence, has become an important public health issue in many developing countries as the treatment of TB requires the use of more expensive drugs for a longer treatment period.

There is, therefore, an urgent need for new, inexpensive TB drugs which are more effective and with less side effects. Medicinal plants are an integral part of South Indian culture.

Centella asiatica Synonyms Hydrocotyle asiatica L. It’s Vernacular name in India: Mandukaparni. Centella has been used as a wound-healing agent and a constituent of a brain tonic for the mentally challenged ⁽⁴⁾. It has also been used traditionally and in Ayurvedic medicine for central nervous system ailments including failing memory, insomnia, depression, stress and epilepsy⁽⁵⁾. In South Africa it was used to treat leprosy, wounds, cancer, fever and syphyllis, while in Europe, the extract has been used for many years to treat wounds. The plant is also used to treat acne and allergies ⁽⁶⁾ and as a psycho-physical regenerator and blood purifier ⁽⁷⁾. The essential oil of Centella showed a broad spectrum of antibacterial activities against Gram-positive (Bacillus subtilis, Staphylococcus aureus) and Gram-negative (Escherichia coli, Pseudomonas aeruginosa, Shigella sonnei) organisms. Activity against Gram-positive bacteria was greater than against Gram-negatives. Germacrene compounds in the essential oil are known to be strong antimicrobial and antitumour agents ⁽⁶⁾. In the present study, an attempt has been made to enrich the knowledge of antimycobacterial activity of Centella asiatica plant extract against Mycobacterium tuberculosis.

MATERIALS AND METHODS:

Anti-Tuberculosis activity:

Lowenstein-Jensen medium base was prepared and sterilized as per the product manufacturer’s instruction. (L-J medium base: 150 ml, beaten egg: 250ml, Glycerol: 3ml) To the prepared L-J media beaten egg was added aseptically and mixed gently without froth. 15 ml medium was aseptically transferred into 30ml McCartney’s culture vial.

The following concentrations of Centella asiatica leaves (10mg, 50mg, 100mg, 200mg, 300 mg, 400mg, 500mg, 600mg, 700mg, 1000mg) ethonolic extract powders were added to (w/v) 15ml of L-J medium and stirred well without froth. Control L-J medium was also prepared without any anti-microbial inhibiting substances. The egg contained L-J medium was coagulated in a slant position at 80⁰C for 15minutes in inspisator.

Inoculum preparation and incubation:

The colonies of Mycobacterium tuberculosis were suspended in 10 ml of sterile normal saline. Vortexed the suspension with sterile glass beads and allowed 30 minutes for larger particles settled down. The upper solution was removed without disturbing the settled particles. The solution was adjusted with sterile normal saline, to a turbidity matching that of a 1.0 McFarland Standard.

From the standard inoculum, 0.05ml of suspension (Himedia: 4mm calibrated nicrome loop was used, Code no: LA 019) was inoculated into the entire L-J medium including control tube. All the L-J media tubes were incubated at 37⁰C for 3-4 weeks. As soon as the completion of incubation the results were recorded as follows.

No growth in antimicrobial containing L-J media: Susceptible

Growth >20 colonies : Partially susceptible

More than 20 colonies : Resistant

Control : Expected to grow >20 colonies

Mycobacterial sensitivity test by Versa- Trek Rapid culture system:

Preparation of Antimicrobial working solution:

The following concentrations of Centella asiatica leaves (10mg, 50mg, 100mg, 200mg, 300 mg, 400mg, 500mg) ethonolic extract powder were added to (w/v) 500μl of sterile normal saline and mixed by vortxeing. (Plate.1)

Preparation of Inoculum:

The colonies of Mycobacterium tuberculosis were suspended in 10 ml of sterile normal saline. Vortxed the suspension with sterile glass beads and allowed 30minutes for larger particles settled down. The upper solution was removed without disturbing the settled particles. The solution was adjusted with sterile normal saline, to a turbidity matching that of a 1.0 McFarland Standard. The standard was further diluted in sterile normal saline with 1:10 ratio.

Anti-mycobacterial assay:^{8; 9}

Antimicrobial substance (500μl), 1ml of growth supplement and 500μl of standard microbial inoculum was aseptically injected into Versa-Trek mycobottle. Like vise all the variable concentrations were inoculated and labeled. The control mycobottle was also prepared with the addition of 500μl sterile normal saline in place antimicrobial substance.

Then the mycobottle was fitted with the connector and loaded into the Versa-Trek rapid culture system with appropriate labeling identification in the touch screen inbuilt computer monitor. The following criteria were used for the growth identification and interpretation of result.

Susceptible: No growth in specific drug for >3 days after the control positivity

Resistant : Growth of Mycobacterium species in specific drug on or before control positivity.

Control : Expected growth positive signals between

3 days to 9 days of incubation

Analysis bioactive compounds in plants by GC MS:

The GC – MS analysis was carried out using a Clarus 500 Perkin–Elmer (Auto system XL) Gas Chromatograph equipped and coupled to a mass detector Turbo mas gold – Perkin Elmer Turbomass 5.1 spectrometer with an Elite – 1 (100% Dimethyl poly siloxane), 30m x 0.25 mm ID x 1µm df capillalary column. The instrument was set to an initial temperature of 110^oC, and maintained at this temperature for 2 min. At the end of this period the oven temperature was rose up to 280^oC, at the rate of an increase of 5^oC/min, and maintained for 9 min. Injection port temperature was ensured as 250^oC and Helium flow rate as 1ml/min. The ionization voltage was 70eV. The samples were injected in split mode as 10:1. Mass spectral scan range was set at 45-450 (m/z).

Using computer searches on a NIST Ver.2.1 MS data library and comparing the spectrum obtained through GC – MS the compounds present in the crude sample were identified.

RESULT AND DISCUSSION:

TB is the leading killer of youths, women, and AIDS patients worldwide⁽¹⁰⁾. Although people with HIV/AIDS are dangerously susceptible to a number of opportunistic infections, TB is without doubt, the main cause of death. There is, therefore, the challenge to fight multidrug-resistant (MDR) TB. People infected with HIV and living with AIDS are at great risk for developing MDR TB. The search for new anti-tuberculosis drugs has become more important due to the above reasons and others such as shortage and expensive nature of TB drugs. Traditional medicine is a readily available alternative in the search for new antimycobacterial compounds. The plants screened in this study are used in traditional medicinal practices for the treatment of various diseases including leprosy and TB.

Plate 1.Centella asiatica Plate. 2 Anti- Tuberculosis activity

Table.1 Mycobacterial susceptibility test of Centella asiatica by Versa-Trek Rapid culture System

Concentration of plant extract	Day of identifiable Growth	Status
10 mg	Growth identified in 10 days	Resistant
50 mg	Growth identified in 10 days	Resistant
100 mg	Growth identified in 11 days	Resistant
200 mg	Growth identified in 12 days	Susceptible
300 mg	Growth identified in 12 days	Susceptible
400 mg	No growth identified for > 16days	Susceptible
500 mg	No growth identified for > 16days	Susceptible
Mycobacterium tuberculosis with no drugs	Growth identified in 8 days	-

Control : Mycobacterium tuberculosis without the given compound

Susceptible: No growth in specific drug for >3 days after control growth positive

Resistant : Growth of Mycobacterium species in specific drug on or before control positive

Table.2 Evaluation of Anti-mycobacterial activity of Centella asiatica Ethanol Extract in LJ medium

Concentration of plant extract	Day of identifiable Growth	Status
10mg	Growth identified within 4 weeks (No of colonies : 5)	Partially Susceptible
50mg	Growth identified within 4 weeks (No of colonies : 4)	Partially Susceptible
100mg	Growth identified within 4 weeks (No of colonies : 3)	Partially Susceptible
200mg	No growth in 4 weeks of incubation	Susceptible
300 mg	No growth in 4 weeks of incubation	Susceptible
400mg	No growth in 4 weeks of incubation	Susceptible
500mg	No growth in 4 weeks of incubation	Susceptible
600mg	No growth in 4 weeks of incubation	Susceptible
700mg	No growth in 4 weeks of incubation	Susceptible
1000mg	No growth in 4 weeks of incubation	Susceptible
Control : M . tuberculosis with no drugs	Growth +ve within 4 weeks of incubation > 20 Colonies	-

In the present study, antimycobacterial activity of ethanol extract of Centella asiatica was evaluated by Versa-Trek rapid culture System. Best antimycobacterial activity was identified by both of the susceptibility methods followed. The lowest concentration of plant extract used inhibits the growth of Mycobacterium tuberculosis is 200mg up to 12 days of incubation time in comparison with control. Table(1).

The antimycobacterial activity of ethanol extract of Centella asiatica was evaluated by LJ medium culture System. The lowest concentration of plant extract used inhibits the growth of Mycobacterium tuberculosis is 200mg within 4 weeks of incubation time in comparison with control (Table 2; Plate 2).

Analysis bioactive compounds in Centella asiatica by GC MS

The Mass spectrum were analysed for crude ethanolic extracts of Centella asiatica showed ten prominent peaks (Fig -1) viz., 9.21 with retention time 1.75 corresponds to copaene peak area ; 13.81 corresponds to tetradecanoic acid with 2.05 peak area; 14.96 corresponds to 3,7,11,15-Tetramethyl-2-hexadacen-1-ol with 4.52; 16.82 corresponds to n- Hexadecadienic acid with 22.69; 19.15 corresponds to 9,12-Octadecadienic acid (Z,Z)- with 32.65; 19.53 corresponds to Octadecanoic acid with 21.77 peak area; 17.14 correspond to hexadecaenoic acid, ethyl ester with 5.52; 17.87 corresponds to 5,8,11-Heptadecatriynoic acid, methyl ester with 3.53 peak area; 19.55 corresponds to 9,12- Octadecadienoic acid (Z,Z)- with 13.52 peak area; 19.67 corresponds to 9,12,15-Octadeatrienoic acid (Z,Z,Z)- with 28.99 peak area; 19.90 corresponds to 9,12,15-Octadeatrienoic acid, methyl ester,(Z,Z,Z)- with 5.67 peak area, 25.94 1,2-Benzenedicarboxylic acid, mono(2-ethylhexyl) ester with corresponds to 3-21% peak areas respectively.(Fig.1; Table.3)

Fig.1 Analysis of bioactive Compounds in Centella asiatica Ethanol plant extract by GC-MS

Table.3 Bioactive Compounds in Centella asiatica Ethanol plant extract

No	RT	Name of the Compound	Molecular Formula	MW	Peak Area %
1	9.21	Copaene	C₁₅H₂₄	204	1.75
2	9.83	Caryophyllene	C₁₅H₂₄	204	0.61
3	10.25	άCaryophyllene	C₁₅H₂₄	204	0.75
4	10.35	1H –Cycloprop[e] azulene,decahydro-1,1,7-trimethyl-4-methylene-,[1aR-(1aά, 4aά, 7ά, 7ά,β,7bά)]-(synonym(+)-Aromadendrene)	C₁₅H₂₄	204	0.75
5	10.63	Phenol,2,5bis(1,1-dimethyl)	C₁₄H₂₂O	206	0.44
6	10.98	Asarone	C₁₂H₁₆O₃	208	1.11
7	11.22	Dodecanoic acid	C₁₂H₂₄O₂	200	1.05
8	13.81	Tetradecanoic acid	C₁₄H₂₈O₂	228	2.05
9	14.96	3,7,11,15-Tetramethyl-2-hexadecen-1-o1	C₂₀H₄₀O	296	4.52
10	16.45	9,12,15-Octadectrienoic acid, methyl ester,(Z,Z,Z)-	C₁₉H₃₂O₂	292	2.34
11	16.82	n-Hexadecanoic acid	C₁₆H₃₂O₂	256	21.77
12	17.14	Hexadecanoic acid,ethyl ester	C₁₈H₃₆O₂	284	5.52
13	17.87	5,8,11-Heptadecatriynoic acid,methyl ester	C₁₈H₂₄O₂	272	3.53
14	18.83	9-Octadecenoic acid(Z)-, methyl ester	C₁₉H₃₆O₂	296	1.27
15	19.20	Phytol	C₂₀H₄₀O	296	1.16
16	19.55	9,12 Octadeca dienoic acid(Z,Z)-,	C₁₈H₃₂O₂	280	13.52
17	19.67	9,12,15- Octadectrienoic acid, (Z,Z,Z)-	C₁₈H₃₀O₂	278	28.99
18	19.90	9,12,15-Octadectrienoic acid, methyl ester,(Z,Z,Z)-	C₁₉H₃₂O₂	292	5.97
19	25.94	1,2-Benzenedicarboxylic acid,mono (2-ethylhexyl) ester	C₁₆H₂₂O₄	278	3.21

The present study is comparable to the findings of Yen et al.,⁽¹¹⁾. Centella asiatica has been used for a long time to treat dermal wounds and leprosy patients in China. Newton et al. ⁽¹²⁾ reported that there were a number of plants, supposedly used in traditional medicine to treat TB, which did not demonstrate any antimycobacterial activity against M. aurum and M. smegmatis on in vitro susceptibility studies. The leaves of the Centella asiatica are traditionally used for to treat TB and other respiratory diseases, it is possible that these plant extracts are effective against ailments such as bronchitis, cough and asthma caused by agents other than Mycobacteria. Anti-mycobacterial screening against TB agent is unsafe because of the highly infectious nature of the pathogen and it is time consuming as the organism is relatively slow growing ⁽¹³⁾. Te current in vitro anti-mycobacterial study of Centella asiatica leaves (ethanolic extract) showed best activities in both Lowenstein Jensen media and with automated Versa-Trek rapid culture susceptibility studies. The results obtained in this study justify the traditional use of Centella asiatica. It possesses potential sources (Octadectrienoic acid and n-Hexadecanoic acid) for the treatment of TB related infections. Currently the new drug discovery is an urgent requirement globally, since there were no new drugs discovered after 1960. The present finding would helpful in controlling the infection, after purification of this vegetative plant product in the development of anti- mycobacterial agents.

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Received on 26.02.2010 Modified on 23.03.2010

Research J. Pharm. and Tech.3 (3): July-Sept. 2010; Page 872-876