Download Antimicrobial, Antitumor and Phytochemical screening for the crude

Antimicrobial, Antitumor and Phytochemical screening for the crude extracts of Leaves, Fruit
flesh and Seed samples of Moringa oliefera
Saminathan ponniaha,b,*, Seethalakshmi Illancheziana
a
Life teck research centre,
No:16/60,Senthil andavar street,
Dhanalkshmi colony,
Vadapalani,
Chennai,
Tamilnadu
India
Pincode:600 026
b
Post graduate department of Microbiology,
Pachaiyappa’s College,
Poonamalle high road,
Chennai,
Tamilnadu,
India
Pincode:600 030
E.mail: [email protected] “* Corresponding author”.
[email protected]
Abstract
Moringa oliefra Lamk. is a member of Moringaceae family and it can be commonly referred to
Moringa. It is an nutritious vegetable tree with a variety of potential use. M.oliefera leaves, seed,
fruit flesh were collected for the present study and the samples were extracted using the solvents
like methanol and petroleum ether. Antibacterial and antifungal study indicated that, the selected
organisms were inhibited by the all the samples but seed extract of methanol showed high level
of inhibition. Minimum inhibitory concentration level for seed extract of both the solvents was
10µg/ml but other samples vary from 10µg/ml to 100µg/ml. The cytotoxic effects of the extracts
on Vero cells and HEp2 cells were assayed using 3-(4,5-Dimethylthiazol-2-yl)-2,5diphenyltetrazolium bromide[MTT] .Phytochemical analysis indicated that all essential
compounds like proteins, flavonids, steroid, sugars were present but in seed sample they may in
high level. Thin Layer Chromatography(TLC) study, Rf value were calculated and seed extract
of methanol showed five bands than leaves and fruit flesh samples.
Key words: M.oliefera, antimicrobial, antitumor, phytochemical ,TLC
1-Introduction:
Moringa oleifera Lamk. is a member of the Moringaceae family. It originated in India,
Srilanka and can be grown up in Asia Minor and Africa as well. Moringa oleifera, commonly
referred to simply as Moringa, is the most widely cultivated variety of the genus Moringa. It is
an exceptionally nutritious vegetable tree with a variety of potential use. Considered one of the
world’s most useful trees, as almost every part of the Moringa tree can be used for food, or has
some other beneficial property. In the tropics it is used as foliage for livestock. The tree has its
origin from the South Indian State of Tamilnadu.
The leaves are highly nutritious, being a significant source of beta-carotene, Vitamin C,
protein, iron , potassium and niazirin [8]. Its leaves are full of medicinal properties. The tree is a
good source for Calcium and Phosphorus21.
In Siddha medicine, the drumstick seeds are used as a sexual virility drug for treating
erectile dysfunction in men and also in women for prolonging sexual activity. The Moringa
seeds yields 38-40 % edible oil (called Ben oil, from high concentration of behenic acid
contained in the oil) that can be used in cooking, cosmetics, and lubrication. Moringa seed
powder is a natural alternative to imported alum (aluminum sulphate, the conventional synthetic
coagulant) used in purifying turbid water in fish culture enclosures (earthen ponds, farm dams
and irrigation canals).
Several evidences revealed that M.oleifera had various pharmaceutical activities such as
antibacterial[5,6], antifungal, antispasmodic, anti-inflammatory, and diuretic activities
[2]
.
Mutagenic activity of M.oleifera was also proposed by Villasena [19]. The nutritional values of
this plant were demonstrated by Chakraborti [3] andVerma [18 ].
Several low molecular weight proteins or peptides with antibacterial or antifungal activity
have been isolated in recent years from various plants
[17]
. Hence, in the present study of
M.oleifera leaf, seed and fruit flesh extracts were taken using methanol and petroleum ether.
Crude extracts of M.oleifera to screen the antibacterial, antifungal, antitumor activity and
phytochemical studies were carried out.
2-Materials and methods:
Fresh leaves, seeds and fruit flesh of Moringa oleifera were collected from Chennai city.
The samples were dried at room temperature and were ground into fine powder with mortar and
pestle. About 5 g of each samples were weighed and added to 100 ml of methanol and 100 ml of
petroleum ether separately and kept at room temperature for 8 hours and filtered with Whatman
No. 1 and evaporated. After evaporation, the crude extract was collected in a eppendorf tube.
This crude extract were used for further study.
2-1-Antibacterial assay:
The cultures of Staphylococcus aureus-MTCC-3160, Bacillus subtilis-MTCC-2274,
E.coli-MTCC-729, Salmonella typhi-MTCC-531 were grown on Brain heart infusion broth (BHI)
for testing antibacterial activity. 0.05mL of the enriched bacterial cultures were added aseptically
to the Mueller Hinton Agar (HiMedia) medium at 450C, mixed well and poured immediately in
sterilized petriplates. After hardening, wells were cut (5mm) with sterile cork borer and 10, 20
and 30 l of the crude extracts were separately added to these wells. The plates were incubated at
37oC for 18 - 24 h. After the incubation period, the diameter of the inhibition zones were
measured [15 ].Experiments were performed in triplicates.
2-2-Antifungal assay:
Antifungal activity was tested against Aspergillus niger-MTCC-514,Trichophyton
rubrum-MTCC-3272 and Candida albicans-MTCC-3958.Potato dextrose agar (HiMedia) was
prepared and 1 mL of crude extract was added to 100 mL of medium and poured immediately in
sterilized petriplates. After solidification a loop full of culture was placed on the centre of the
plate. Controls were maintained with DMSO only. All the plates were incubated at 28oC for1-5
days. The growth of the fungal cultures were measured from the date of incubation to till the last
day and compared with the respective control plates. Experiments were performed in triplicates.
2-3-Determination of minimal inhibitory concentration (MIC):
The minimum inhibitory concentration was determined by serial dilution in the same type
of agar, with concentration of extracts ranging from 10, 50 and 100 g/ml. The inoculum was
prepared from fresh overnight broth cultures. Plates were incubated for 24 hours at 370C and 96
hours at 280C for antibacterial and antifungal activity respectively. Further studies the methanol
and petroleum ether extract of seed sample was carried out.
2-4-Cell Viability Assay:
Vero cells (African green monkey kidney cells)obtained from King Institute of
Preventive Medicine, Chennai, India, was used to determine the non-toxic dose of the extracts of
seed samples. The cells were grown in a 24- well plate in eagle’s minimum Essential Medium
(HiMedia)
supplemented
with
10%
fetal
bovine
serum
(Gibco
Laboratories)
and
antibiotics(streptomycin, penicillin-G, kanamycin, amphotericin B, HiMedia). About 1 mL cell
suspension (105 cells mL) was seeded in each well and incubated at 370C for 48 hrs in 5% CO2
for the formation of confluent monolayer. The monolayer of cells in the plate was exposed to
various dilutions of the extracts. The cell viability was measured using MTT assay [11] with MTT
(5mg/ML) and DMSO. This tetrazolium salt is metabolically reduced by viable cells to yield a
blue formosan product measured at 540nm Spectrophotometerically
[11]
. Controls were
maintained throught the experiment (Untreated wells as cell control and diluent treated wells as
diluent control). The assay was performed in triplicate for each of the extracts. The mean of the
cell viability values was compared to the control to determine the effect of the extract on cells
and % cell viability (Vero cells) was plotted against concentration of the plant extract.
2-5-Antitumor assay:
The antitumor assay was performed on Human laryngeal epithiloma cells(HEp2) cells
obtained from King Institute of Preventive Medicine, Chennai, India. With non-toxic dose of the
extracts of seed samples and its dilutions. The cell viability was measured using MTT assay as
describes above. Controls were maintained thought the experiment (Untreated wells as cell
control and diluent treated wells as diluent control) The assay was performed in triplicate for
each of the extracts. The minimum concentration of plant extract that was non-toxic to vero cells
but toxic to HEp2 cells was recorded as the effective drug concentration.
2-6-Preliminary phytochemical analysis
Phytochemical screening was carried out by the method described by Sofowora
[16]
.Screening were carried out on steroid, sugar, tannin, protein, flavanoid, saponin, terpenoid and
alkaloid.
2-7-Thin Layer Chromatogram Analysis
The slurry was coated over a glass plate at a thickness of 0.25 mm. The plates were kept
in an oven between 100ºC or 250ºC for the activation of the adsorbent. Spot of the crude
methanolic and petroleum ether samples were done about 1.5cm from the bottom of the plate by
using capillary Tube. The developing solvent (Toluene: ethylacetate : formic acid, 7:3:1) was
poured into the tank with a depth of 1.5cm. When the solvent had reached the top of the plate. It
was removed from the tank, dried and the spots were seen on illuminator. The green and yellow
colour spots were developed and RF values were calculated as Rf= Distance traveled by the
solute/ Distance traveled by the solvent.
3-Results:
The results of antibacterial, antifungal, antitumor ,phytochemical , TLC from methanol
and petroleum ether extracts of M.oleifera leaves, seed, fruit flesh were shown in Table 1to6.
Antibacterial activity of methanol extract from Seed showed maximum activity against S.
aureus, E. coli, S. typhi and B. subtilis, when compared to the Fruit Flesh and Leaves extracts.
Petroleum ether extract from Seed also showed more activity against S. aureus and E. coli
similar to that of methanol seed extract .For fruit flesh extract the inhibition level for all the
bacteria between 5 – 10 mm, but for leaves extract the inhibition level for all the bacteria
between 10 -14 mm. The present study indicated that both methanol and petroleum ether extract
of M.oleifera seed was more active than fruit flesh and leaves extracts.
Antifungal activity of methanol extract of seed showed high inhibition against
Trichophyton rubrum and Aspergillus niger than Candida albicans . However fruit flesh and
leaves extracts showed only low inhibition. Petroleum ether extract of seed also found more
inhibition against Trichophyton rubrum and Aspergillus niger. Fruit flesh extract showed
inhibition level for all the fungal between 15 – 20 mm, but for leaves extract the inhibition level
for all the fungal species was between
21 -25mm. It indicates that both crude extracts taken
from methanol and petroleum ether of M.oleifera seed showed more inhibition level.
For cell viability assay, both the extracts showed the highest cytotoxic activity on Vero
cells. MTT assay was used to evaluate cytotoxicity based on metabolic reduction of MTT. On
treatment with Vero cells. 1:2 and 1:4 dilutions of both the extracts showed cytotoxicity but there
was no apparent cytotoxicity at 1:8 dilution. Further dilutions also had no toxic effects on Vero
cells. But for antitumor assay, methanol extracts showed the highest cytotoxicity than petroleum
ether on HEp2 cells. 1:8 and 1:16 dilutions of the methanol extract showed cytotoxicity and
further dilutions also had less effect on the viability of the cancerous cells.1:8 dilution of the
petroleum ether extract showed cytotoxicity and further dilutions also had less effect. Thus, the
1:8 dilution of the methanol extract of M.oliefera is non-toxic to the normal cells and also had
anticancer activity against the cancerous cells.
Phytochemical screening of M. oleifera (Leaves, Fruit Flesh, and Seed) was recorded.
Result showed that both the extracts contain Steroid, Sugar, Protein and Flavonoid. But both the
extracts, Tannin, Saponin and Alkaloids were absent. In petroleum ether extract Terpenoids also
present but it was absent in methanol extract. Our results revealed that the presence of
phytochemical parameters were similar to all the samples like Seed, Flesh and Leaves of
methanol and petroleum ether extracts
For the TLC analysis, no bands were seen in petroleum ether extract but 3 to5 bands were
obtained in methanol extracts and their Rf value were calculated and tabulated.
5-Discussion:
In the present study of M. oleifera crude extract samples were collected from seed, fruit
flesh and leaves and tested against bacteria, fungi and tumor cell study were determined. The
crude extract of leaves, seed and fruit flesh taken in methanol fraction had showed maximum
antifungal and antibacterial activity .However moderate activity showed by petroleum ether
fraction.
It is clearly indicated that methanolic extract of M. oleifera leaves, fruit flesh and seed
poses significant antimicrobial activity against gram-positive and negative bacteria and fungus
studied. The similar results was obtained in the study of Nantachit [9], the capsules, leaves and
seeds of M.oleifera have high nutritional value, vitamins and many minerals. The seeds of this
plant also showed antimicrobial activity and it found that against gram-positive and gramnegative bacteria. Moringa leaf extracts exhibited antimicrobial activity including inhibition of
the growth of Staphylococcus aureus strains isolated from food and animal intestines
[12]
. Our
results also proved that leaf extract of both the solvent showed the high level of inhibition on
Staphylococcus aureus.
To our knowledge this is the first time Moringa fruit flesh has been tested for
antimicrobial activity. The crude extracts at some concentrations showed on activity probably
there was not enough active constituent in the crude extract. Various evidences revealed that M.
oleifera had several pharmaceutical activities [6], antifungal, antiplasmodic, anti-inflammatory
and antidiuretic activities
[2]
. This observation provides strong circumstantial evidence that
fraction presents in the plant play an important role in plants defence system
M.oleifera was found among the most promising species according to their high
antioxidant activity, high contents of micronutrients and phytochemicals. For phytochemical
screening, tannins was absent in all the extracts of leaves, seed and fruit flesh samples. Tannins
with its protein precipitating and vasoconstriction effect could be advantageous in preventing
ulcer development. Presence of flavonoids have been reported to offer some protection in ulcer
development by increasing capillary resistance, and improve microcirculation which renders the
cells injurious to precipitating factors[1].The presence of proteins in all the samples of M. oleifera
is a good source of essential amino acids. This agreed with Ram [13] reported that, M. oleifera
seeds contained all the essential amino acids in appreciable quantities. This implies that, all these
plants can equally serve as good sources of amino acids for man and livestock.
The antibacterial, antifungal and antitumor activity was at its peak in methanolic extract
indicating that most of the active components are extracted with methanol. The overall results
indicate promising baseline information for the potential uses of the methanol extract of M.
oliefera seed than fruit flesh and leaves in the treatment of infectious diseases and tumor. Further
work should be carried out to purify and possible characterize the active principle responsible for
the observed effect. Also additional work should be embarked upon with a view to elucidate the
possible mechanism of action of the extract.
6-References:
[1 ] Caceres,A.B; Cabrera O; Mirals, O. Mollinedo,O. and Imendia, A.1991. Preliminary
screening for antimicrobial activity of Moringa oleifera. J.Enhnopharmacol.33:213216.
[2] Caceres, A., A.Sarawia, S. Rizzo, L. Zabala, E. De Lean, and F. Nove. 1992.
Pharmalocogical properties of Moringa oleifera II: Screening for antispasmodic, antiinflammatory and diuretic activity. Journal of Ethnopharmacology 36(3): 233-237.
[3] Chakraborti, N., L.Mordal, and G.C.Banerjee. 1988. Chemical composition of some
common tree leaves. Indian Vet.J.65 (2): 145-149.
[4] Dahiru,D., Onubiyi,J.A. and Umaru,H.A.2006.Phytochemical screening and
Antiulcerogenic effect of Moringa oleifera aqueous leaf extract.3:3:pp.70-75
[5] Dayrit, F.M., A.D.Angela and I.M. Villasena.1990.Studies on Moringa oliefera seeds.
Part I. The antibiotic compound and its deactivation in aqueous solutions.
Phillipp.J.Sci.119(1):23-32
[6] Eilert, U., B Walters, and A.Nahrstedt.1981. The antibiotic Principle of seeds of M.
oleifera and Moringa stenopatala. Planta Med.42(1):55-61.
[7] Grabow,W.O.K., Slabbert,J.L., Morgan,W.S.G. and Jahn, S.A.A. 1985. Toxicity and
mutagenicity evaluation of water coagulated with Moringa oleifera seed preparation using
fish protozoan, bacterial coliphage, enzyme and salmonella assay. CAB Abstract 1984-1986.
[8] Karuna Shanker, Madan M. Gupta, Santosh K. Srivastava. 2007. Central Institute of
Medicinal and Aromatic Plants, PO- CIMAP, Lucknow 226 015, India.
[9] Khesorn Nantachit.2006.Antibacterial activity of the capsules of Moringa olifera
Lamk.(Moringaceae)CMU Journal.Vol.5(3):365-368.
[10] Linday EM. 1962. Practical Introduction to Microbiology, E&FN Spon Ltd., pp 77.
[11] Mosmann T. 1983.Rapid colorimetric assay for cellular growth and survival:
application to proliferation and cytotoxicity assays. J Immunol Methods. 65:55-63.
[12] Ray-Yu Yang, Lien-Chung Chang, Jenn-Chung Hsu,Brain B.C.Weng, manuel
C.Palada, M.L.Chadha and Virginie Levasseur.2006.Nutitional and Functional Properties of
Moringa Leaves- From Germplasm, to Plant, to Food, to Health.Moringa and other highly
nutritious plant resources:Strategies,standardsand markets for a better impact on nutrition in
Africa,Accra, Ghana:16-18.
[13]Ram, J., 1994, Moringa: A highly nutritious vegetable tree. Triades Technical
bulletin number 2
[14] Ramachandran ,C., Peter KV.and Gopalakrishnan P.K., 1980, Drumstik(Moringa
oleifera): A multipurpose Indian Vegetable. Economic Botany, 34 (3) pp 276-283.
[15] Rashed A, Khan MR, and Khalid N: 1990.Antibacterial activity of some medicinal
plants and seeds. Pak J Biochem,23:55-62.
[16] Sofowora, E.A.1994.Medical Plant and Traditional Medicine in Africa. University
of Ife Press, Nigeria: pp.1-23
[17] Terras FRG,Schoofs HME, De Bolle MFC, Van Leven F, Rees SB, Vanderieyden J,
Cammue BPA and Broekaert WF: 1992.Analysis of two novel classes of antifungal
proteins from radish (Raphanus sativaus)seeds. J.Biol Chem 267:15301-15309.
[18] Verma,S.C., R.Banerji,G.Mirsa, and S.K.Nigam. 1976.Nutritional value of Moringa.
Curr.sci.45(21):769-770.
[19] Villasena,I.M., P.Finch, C.Y.Lim-Sylianco, and F.Dayrit. 1989. Structure of a
mutagen from roasted seed of Moringa oliefera. Carcinogenesis(London).10(6):10851088.
[20]Umar Dahot M, 1998, Antimcrobial activity of small protein of Moringa oleifera
leaves. Journal of Islamic academy of Sciences 11:1, 27-32.
[21] Yang R.Y., Tsou, S.C.S., Lee, T.C., Chang ,L.C., Kuo,G., and Lai,P.Y. 2006. Moringa,
a novel plant rich in antioxidants, bioavailable iron, and nutrients. pp224-239.In:
C.T. Ho(ed) Challenges in Chemistry and Biology of Herbs.
Acknowledgments:
Authors wish to thank to Director, Life teck research centre, Vadapalani, Chennai, Tamilnadu,
India for providing of facilities and motivation.We also put forward our sincere thanks to
Principal, Pachaiyappa’s college, Chennai, Tamilnadu, India, for his encouragement.
Tables: 1 to 6:
Table –1
Antibacterial activity of methanol and petroleum ether extracts of Moringa oleifera seed, fruit
flesh and leaves.
Seed extracts
S. aureus
Petroleum
Methanol (µl)
Organisms
Fruit Flesh extracts
ether (µl)
Leaves extracts
Petroleum
Methanol (µl)
ether (µl)
Petroleum
Methanol (µl)
ether (µl)
10
20
30
10
20
30
10
20
30
10
20
30
10
20
30
10
20
30
+++
+++
+++
++
++
+++
+
+
+
+
+
+
+
+
++
++
++
++
+++
+++
+++
+++
+++
+++
+
+
+
+
+
+
+
++
++
+
++
++
++
+++
+++
++
++
+++
+
+
+
+
+
+
+
+
++
++
++
++
++
++
++
++
++
++
+
+
+
+
+
+
+
++
++
++
++
++
MTCC-3160
E. coli
MTCC-729
S.typhi
MTCC-531
B.subtilis
MTCC-2274
( + ) = 5 to 10 mm inhibitory zone; (++) = 10 to 14 mm inhibitory zone; (+++) = 15 to 20 mm inhibitory zone
Table – 2: Antifungal activity of crude extract taken from seed, fruit flesh and leaves:
Organism
Seed extract
Methanol
Petroleum
Fruit Flesh extract
Methanol
Petroleum
ether
Leaves extract
Methanol
Petroleum
ether
ether
C.albicans
MTCC-3958
1st day
2nd day
3rdday
4th day
5th day
T. rubrum
+
+
+
+
++
+
+
++
++
++
+
+++
+++
++++
++++
+
++
++
+++
+++
+
+++
+++
++++
++++
+
+
+++
+++
++++
+
++
+++
+++
+++
+
++
++++
++++
++++
+
+
++
++
++
+
+
++
++
++
+
++
+++
+++
+++
+
++
++
++
++
++
++
+++
+++
+++
+
++
++++
++++
++++
+
+++
+++
+++
++++
++
++
++
++
++
+
++
++
++
++
++
++
+++
++++
++++
MTCC-3272
1st day
2nd day
3rdday
4th day
5th day
A. niger MTCC-514
1st day
2nd day
3rdday
4th day
5th day
(+ )
= 5 to 14 mm inhibitory zone;
(++) = 15 to 20 mm inhibitory zone,
(+++)
= 21 to 25 mm inhibitory zone;
(++++)= 26 to 30 mm inhibitory zone.
Table:3: Minimal inhibitory concentration crude extract taken from seed, fruit flesh
and leaves of M.oleifera.
Test organism
Seed Extracts
Methanol
(g/ml)
10 50 100
Bacteria
Staphylococcus
aureus
+
-MTCC-3160
E.coli-MTCC729
+
Salmonella
typhiMTCC-531
Bacillus
subtilisMTCC-2274
Fungi
Candida
albicansMTCC-3958
Trichophyton
rubrumMTCC-3272
Aspergillus
nigerMTCC-514
Fruit Flesh Extracts
Leaves Extracts
Petroleum
ether(g/ml)
10 50 100
Methanol
(g/ml)
10 50 100
Petroleum
ether(g/ml)
10 50 100
Methanol
(g/ml)
10 50 100
Petroleum
ether(g/ml)
10 50 100
+
+
+
+
+
-
-
+
-
-
+
-
-
+
-
-
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
-
-
+
-
+
+
-
-
+
-
-
+
-
-
+
+
+
+
-
-
+
-
-
+
-
-
+
-
+
+
-
-
+
+
+
+
-
-
+
-
-
+
-
-
-
-
-
+
-
-
-
+
+
+
-
-
+
-
-
-
-
-
-
-
-
-
-
-
-
+
+
+
-
+
+
-
+
+
-
-
-
-
-
-
-
-
-
(+) Presence of inhibition ; (-) Absence of inhibition
Table:4: Anti cancer assay(Hep2) of Moringa oleifera extracts by cytotoxic assay
Plant
Plant Extracts
Methanol
M.oleifera
Petroleum Ether
Dilutions
1:8
Toxicity
1:16
1+
1:32
0
1:64
0
1:8
1+
1:16
0
1:32
0
1:64
0
1+
Table-5; The phytochemical profile of leaves, fruit flesh and seed showed various compounds.
S.No
Phytochemical
Methanol
tested
L
F.F
1.
Steroid
+
-
2.
Sugar
+
3.
Tannin
4.
Petroleum ether
S
L
F.F
S
-
+
+
-
+
+
+
+
+
+
-
-
+
-
-
Protein
+
+
+
+
+
+
5.
Flavanoid
+
-
-
+
+
-
6.
Saponin
-
-
+
-
-
+
7.
Terpenoid
+
-
-
+
-
+
8.
Alkaloid
+
-
-
-
-
-
L- Leaf; F.F- Fruit Flesh; S- Seed; (+) = Presence; (-) = Absence
Table:6 : Rf values of M. oleifera methanol extracts of seed, fruit flesh, leaves were
tabulated.
Flesh
Seed
Leaves
0.58
0.54
0.58
0.41
0.44
0.23
0.32
0.32
-
-
0.17
-
-
0.08
-