Mycobacterium tuberculosis, the infective agent of tuberculosis(TB) Causes one of the world’s most devastating global health crisis The treatment of drug-susceptible TB lasts more than six-months and require the use of drugs to achieve a cure, whereas for XDR treatment lasts for more than1 year The two most effective first- line TB drugs for drug susceptible TB are isoniazid (INH) and rifampicin(RIF), which has to be taken for six months. The long treatment period is of major concern as it leads to the development of the drug resistant TB. The study found that anti-tuberculosis drugs killed more bacteria in laboratory when the mice received vitamin C.

1. EXPLORING THE MYCOBACTERICIDAL POTENTIAL OF VITAMIN C RICH FRUITS
GROWING IN JAMMU REGION
MASTER OF SCIENCE
IN
MICROBIOLOGY
SCHOOL OF BIOTECHNOLOGY
UNIVERSITY OF JAMMU
JAMMU-180006
PRESENTED BY:
TARUN SAROCH(03-MBL-16)

2. CONTENTS
• INTRODUCTION
• OBJECTIVE
• METHODS
• RESULTS
• DISCUSSION
• CONCLUSION
• REFERENCES

3. INTRODUCTION
•Mycobacterium tuberculosis, the infective agent of tuberculosis(TB)
Causes one of the world’s most devastating global health crisis
•The treatment of drug-susceptible TB lasts more than six-months and
require the use of drugs to achieve a cure, whereas for XDR treatment
lasts for more than1 year
•The two most effective first- line TB drugs for drug susceptible TB are
isoniazid (INH) and rifampicin(RIF), which has to be taken for six months.
•The long treatment period is of major concern as it leads to the
development of the drug resistant TB
•The study found that anti-tuberculosis drugs killed more bacteria in
laboratory when the mice received vitamin C.

4. •Vitamin C has been found to show significant bactericidal activity and it
aids to decrease the time period of the treatment and also limits the
occurence of drug resistance (vilcheze et al. 2013)
•It has been previously demonstrated that high concentration of vitamin C
sterilizes drug-susceptible, MDR and XDR M.tuberculosis cultures and
prevent the emergence of drug-persisters(vilcheze C et al.,2018)
•The benefit of plant derived medicine to reduce side effects that are often
associated with synthetic drugs is well known.
•Many Plants are traditionally used as remedies to cure various
Respiratory Diseases.
•So we decided to check the antimycobacterial activity of some locally
grown plants which are rich in vitamin C.

5. • Hydroxyl radicals induce cell death via DNA damage. Hydroxyl
radicals can be produced by the combination of the Haber-Weiss
cycle and the Fenton reaction. ( Foti JJ, Haber F, Fenton H et al.,
2012,1934,1894).
• Fe2+
+ O2 → Fe3+
+ O2
•−
(1)
• 2O2
•−
+ 2H+
→ H2O2 + O2
(2)
• H2O2 + Fe2+
→ Fe3+
+ OH•
+ OH−
(3)
• In the presence of reductants, ferrous ions are produced by
reduction of ferric ions. One reductant known to drive the Fenton
reaction is vitamin C (Burkitt MJ et al., 1990).

6. Fenton and haber-weiss reaction

7. OBJECTIVE
• To Check the antimycobacterial/killing
potential of fruits growing in Jammu region
which are rich in vitamin C by using
microtitre plate assay method.

8. MATERIAL AND METHODS

9. MATERIAL
Selection of plants: The plants which have high vitamin C were selected, by a
thorough literature survey.

10. • Plants were chosen keeping in mind their high concentration of vitamin C
and which were easily available in Jammu(J&K) based on literature.
We selected 3 different plants namely Ziziphus mauritiana (Ber), Rosa
(Rose hip) Myrciaria dubia (Camu camu),
Ziziphus mauritiana (ber)
(Rhamnaceae)

11. Myrciaria dubia (Camu camu)
(Myrtaceae)

12. Rosa(Rose hip)
(Rosaceae)

13. •Plants fruits were collected between spring and summer season during
March to May 2018.
•The healthy parts were surface sterilized with 70% ethanol.
•Then 3%MPA extracts of the fruits were made to keep vitamin C stable
•REAGENTS USED:
I. Stabilizing medium:
• (3%w/v)15 g of metaphosporic acid(HPO3), stir after adding 50ml of
distilled water and make up the volume to 500ml and keep it in cold.

14. II. 2,6-dichlorophenol indophenol dye solution: 125mg of sodium salt of dye
dissolved in 105mg NaHCO3 in 100ml hot distilled water(about 80˚C).Cool,
filter and make up the volume to 500ml with distilled water. Always prepare
fresh.
III. Standard ascorbic acid solution(1mg/ml): 100mg of ascorbic acid in 100ml of
distilled water
• BACTERIAL STRAINS AND REAGENTS:-
• The M.tuberculosis H37Rv strain used in the study was obtained from
laboratory stocks and was grown at 37 C in Middlebrook 7H9
medium suplemented with (10% OADC enrichment (oleic acid-
albumin-dextrose catalase, 0.2% glycerol and 0.05% tyolaxapol.

15. METHODS

16. 1.
•Weigh approx 10g of sample in stabilizing medium(1:5) in
mortar pestle.
•Centrifuge the homogenate
2.
• Take the supernatant and make up the volume to 100ml
with stabilizing medium
• Filter through Whatman No.1 filter paper
3.
• After that samples were dried at room temperature
for 2-3 days for further experiments.
PREPARATION OF 3% MPA EXTRACT OF SAMPLE AND VITAMIN C
QUANTIFICATION

17. STANDARDIZATION OF DYE
• 1ml of standard ascorbic acid(1mg/ml)in 50ml conical flask and titrate with
2,6-dichlorophenol indophenol dye solution to faint pink colour, which should
persist for atleast 15 sec.
• Note the titre value.
Estimation of ascorbic acid in collected samples:
• Then known volume(5-10ml) of m-phosphoric acid extract of sample in 50ml
of conical flask and titrate with standard dye to faint pink colour which should
persist for 15 sec.

18. CALCULATION:
Amount of ascorbic acid(mg/ml):
= X V2 Z
x x x 100
________ _________ ___________
V 1 Y Wt. of sample
X = mg of standard ascorbic acid
V1 = Titre value of standard ascorbic acid against dye
V2 = Titre value of sample against dye
Y = Amount of aliquot taken(1ml) for estimation
Z = Total volume made up of extraction sample

19. EXTRACT OF PLANTS (FRUITS)
3% MPA Rosehip extract 3%MPA Camu camu extract 3%MPA Ber extract

20. 1 27 100
______________ X __________ X _________ X 100 = 486 mg/100g of vitamin C in BER
10.7 5 10
S.NO INITIAL
READING
FINAL
READING
INITIAL - FINAL
1 0 26.5 26.5
2 26.5 54.5 27
3 54 81 27
CONCORDANT READING = 27

21. • 2371.5mg/100g of vitamin C in CAMU CAMU
S.NO INITIAL
READING
FINAL READING INITIAL - FINAL
1 0 52.7 52.7
2 52.7 104.4 51.7
3 104.4 210.8 52.7
Concordant reading = 52.7

22. • 1881mg/100g of vitamin C in ROSE HIP
S.NO INITIAL READING FINAL READING INITIAL - FINAL
1 0 3.8 3.8
2 3.8 7.6 3.8
3 7.6 11.4 3.8
Concordant reading = 3.8

23. INOCULUM PREPARATION AND
DETERMINATION OF MBC
Mycobacterium tuberculosis H37Rv (ATCC 25177) was used. The cultures
were maintained on Lowenstein-Jensen media slant at 37 °C.
•Stock of plant extract was made of a concentration equals to 10mg/ml
•Then 100µl of 256µg/ml extracts was added to the 2nd column and then it
was serially diluted to make the concentrations from 128µg/ml till
0.125µg/ml
•Then we took control drug rifampicin from a starting concentration of
2µg/ml and diluted till 0.0039µg/ml.
•10 mcFarland unit of culture of mycobacteria H37RV was added in each
well.
•The plates were incubated for 8 days and then 25µl dye resazurin was
added, plates were sealed and further re-incubated for 6 days to check
MBC

24. REPRESENTATION OF MICROTITRE PLATE
MICROTITRE PLATE DATA
ROWS
A= WATER
B= camu camu extract
C= Rosehip extract
D= Ber extract
E= empty
F= POSITIVE CONTROL (RIFAMPICIN)
G, Column 11 = -ve control

25. DETERMINATION OF MBC OF DRUG WHEN GIVEN IN
COMBINATION WITH PLANT EXTRACT
• For checking the mbc of drug when given in
combination with extracts, we took extracts of rosehip,
camu camu and ber in of concentrations 64µg/ml and
32µg/ml and 128µg/ml respectively.
• Drug rifampicin was serially diluted from 2µg/ml till
0.003µg/ml.
• 10 mcFarland unit of culture of mycobacteria H37RV
was added in each well.
• The plates were incubated for 8 days and then 25µl
dye resazurin was added, plates were sealed and
further reincubated for 6 days to check MBC.

26. RESULTS
Minimum Bactericidal concentration of the extracts
S.NO EXTRACTS MBC Of extracts alone (µg/ml) MBC( given in combination with
drug rifampicin)
1 ROSEHIP (A) 64 0.03
2 JUJUBE (B) >128 0.06
3 CAMU CAMU (C) 32 0.03
4 CONTROL 0.06 0.06
PICTURE FOR RERPRESENTATION PURPOSE ONLY
SOURCE: ( Coban et. al 2012)

27. Mycobactericidal activity of rosehip
compared to control
0.015625
0.03125
0.0625
0.125
0.25
0.5
1
2
4
8
16
32
64
128
ROSEHIP CONTROL
DILUTIONCONCENTRATIONS(µg/ml)
MBC
ROSEHIP
CONTROL
1. Graph showing MBC concentration of 3% MPA extract of Rose hip

28. Mycobactericidal activity of camu
camu compared to control
• 2. Graph showing MBC concentration of 3% MPA extract of Camu Camu
0.015625
0.03125
0.0625
0.125
0.25
0.5
1
2
4
8
16
32
64
128
CAMU CAMU contol
MBC
CAMU CAMU
contol

29. Mycobactericidal activity of Indian
Jujube compared to control
3. Graph showing MBC concentration of 3% MPA extract of Indian jujube
0.015625
0.03125
0.0625
0.125
0.25
0.5
1
2
4
8
16
32
64
128
BER CONTROL
MBC
BER
CONTROL

30. Mycobactericidal activity of drug when given in
combination with extracts
0
0.01
0.02
0.03
0.04
0.05
0.06
0.07
0.08
0.09
0.1
CONTROL Rifampicin+Rosehip Rifampicin+Ber Rifampicin+ Camu
Camu
concentrationinµg/ml
Exracts in combination with drug (Rifampicin)
MBC
CONTROL
Rifampicin+Rosehip
Rifampicin+Ber
Rifampicin+ Camu Camu

31. DISCUSSION
• Vitamin C is known to be an anti-oxidant and a pro-oxidant. The
sterilizing effect of vitamin C on M. tuberculosis cultures is most
likely due to it s pro-oxidant activity.( Podmore ID, et al., Frei B et al.,
1998-89)
• In a strain of M. tuberculosis that does not produce mycothiol, the
bactericidal effect of vitamin C is increased sixty fold compared to a
wild-type strain, resulting in the killing of 107 cells in a week.(Newton
GL , et al., Metaferia BB, et al., 2011,2007)
• In an earlier study, terminally ill TB patients were given daily high
doses (15 grams/day) of vitamin C orally for 6–8 months and
showed no side effects. Although the author did not observe any
regression of the TB lesions, the effects on the 130 bedridden
patients were described as remarkable; they had regained appetite
and physical activity (Charpy J et al., 1948)

32. •A study showed that TB patients who received a daily supplement of
vitamin C with their routine diet and treatment had a better haematological
response than the TB patients who did not get the vitamin C supplement,
suggesting a better prognosis, even though at that time, none of the first-
line TB drugs were available and treatment options were limited. (Rudra
MN et al., 1946)
•M.tuberculosis infected mice co-treated with vitamin C (1 g/kg, daily
subcutaneous injection) and INH (25 mg/kg, daily subcutaneous injection)
for 74 days were found to have no sign of infection at the end of treatment
whereas the mice treated with INH had visible lesions. (Venulet F et
al.,1955)

33. CONCLUSION
• The maximum MBC was shown by CAMU CAMU which is equal to
32µg/ml and ROSE HIP also showed very good MBC i.e 64µg/ml
• Indian jujube showed the minimum MBC among all.
• But when extracts were given in combination with drug, they
decreased the MBC of drug i.e significantly enhanced its efficacy .
• When given in combination with camu camu, the MBC of RIF
decreased from 0.06 to 0.03µg/ml and same results were seen in case
of Rosehip.

34. FUTURE PROSPECTIVES
•The addition of fruit extract of vitamin C to TB chemotherapy
might shorten TB treatment, leading to a reduction in the
incidences of both drug resistance development and disease
relapse.
•Also including these locally available fruits in diet can boost
the immune system and make an individual less susceptible
to the infection

35. REFERENCES
•PodmoreID,etal. Vitamin C exhibits pro-oxidant
properties. Nature. 1998;392:559. [PubMed]
• Frei B, England L, Ames BN. Ascorbate is an outstanding antioxidant in
human blood plasma.Proc.Natl.Acad.Sci.UA.[PMC free article] [PubMed]
•Newton GL, Buchmeier N, La Clair JJ, Fahey RC. Evaluation of
NTF1836 as an inhibitor of the mycothiol biosynthetic enzyme MshC in
growing and non-replicating Mycobacterium tuberculosis. Bioorg. Med.
Chem. 2011;19:3956–3964. [PMC free article] [PubMed]
• Metaferia BB, et al. Synthesis of natural product-inspired inhibitors
of Mycobacterium tuberculosismycothiol-associated enzymes: the first
inhibitors of GlcNAc-Ins deacetylase. J. Med. Chem. 2007;50:6326–
6336. [PubMed]

36. • Taneja NK, Dhingra S, Mittal A, Naresh M, Tyagi JS. Mycobacterium
tuberculosis transcriptional adaptation, growth arrest and dormancy
phenotype development is triggered by vitamin C. PLoS
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administrée seule ou associée à la vitamine D2, dans la tuberculose.
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tuberculosis and the effect upon it of large doses of vitamin C. Tubercle
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tuberculosis is extraordinarily sensitive to killing by a vitamin C-induced
Fenton reaction. Nat. Commun. 4:1881.
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37. THANK YOU