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INTERNATIONAL JOURNAL OF PHYTOTHEARPY RESEARCH 
ISSN 2278 – 5701 
Original Research Article 
POTENTIAL USES OF Moringa oleifera AND AN 
EXAMINATION OF ANTIBIOTIC EFFICACY CONFERRED BY 
M. oleifera SEED AND LEAF EXTRACTS USING CRUDE 
EXTRACTION TECHNIQUES AVAILABLE TO 
UNDERSERVED INDIGENOUS POPULATIONS 
Rockwood, J.L.1*,Anderson, B.G.2*,Casamatta, D.A.3 
1 MS4, The University of Texas Health Science Center, San Antonio, TX, USA 
2 DO, Lake Erie College of Osteopathic Medicine, Erie, PA, USA 
3 PhD, University of North Florida, Jacksonville, FL, USA 
ABSTRACT: 
Moringa oleifera, a pantropical plant, is one of approximately thirteen species belonging to the monogeneric 
Moringaceae family. Ethnobotanical studies conducted in Guatemala found that one of the primary medicinal 
purposes of M. oleifera was its use for the treatment of infectious skin and mucosal diseases. As it is common 
practice for researchers to scientifically validate the efficacy of traditional medicine, it is less common for 
researchers to scientifically validate simple, reproducible means of conferring therapeutic benefits of plant parts. 
This study was conducted to investigate pragmatic extraction techniques for seed and leaf extracts of M. oleifera, a 
plant species for which numerous studies have demonstrated its antimicrobial efficacy. M. oleifera seeds and leaves 
were extracted using three different solvents (de-ionized water, inorganic ethanol, organic ethyl acetate) and two 
different extraction methods (crude, sophisticated). Sensitivity disks impregnated with the various extracts were 
used for antibiotic susceptibility testing of fourteen bacterial species: seven representative Gram-negative and seven 
representative Gram-positive. De-ionized water was the only solvent capable of extracting plant constituents which 
conferred bacterial inhibition. Seed extracts were found to inhibit a broader range of organisms (4) than leaf extracts 
(1). 75% of the organisms inhibited by seed extracts were Gram-positive bacteria. A single parameter, the zone of 
inhibition, was used to compare antibacterial efficacy between extraction methods, trials, and controls. No 
difference was observed between the zone of inhibition of crude and sophisticated extracts. Seed extracts 
demonstrated a zone of inhibition comparable to that of penicillin and tetracycline. 
Keywords:Moringa oleifera,Crude extraction,Phytomedicine 
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INTRODUCTION: 
Antibiotic drugs are one of the many tools in 
our current arsenal of medical defense. 
Penicillin is a prime example of how 
modern antibiotics revolutionized our 
capacity to combat morbidity and mortality 
resulting from bacterial infections. Despite 
great strides and advances in medical 
equipment and therapies, infectious diseases 
have long remained the leading cause of 
morbidity and mortality in the developing 
world [1]. A sober approach to examining 
the underlying cause for such disparities 
requires acknowledging that within the 
current climate of market-driven economies 
and for-profit pharmaceutical practices, 
people often suffer from curable and 
preventable diseases due to the expenses 
associated with research, development, and 
delivery of healthcare [2]. 
Resources elucidating current research 
efforts and statistical data for the ‘big-three’ 
communicable diseases – malaria (resulting 
from infection with Plasmodium species), 
AIDS (resulting from infection with HIV), 
and tuberculosis (resulting from infection 
with Mycobacterium species) - abound the 
annals of scientific literature. Nevertheless, 
documentation of research aimed toward 
pragmatic and reproducible antibiotic 
therapies for residents of resource-poor 
settings is comparatively small. According 
to Osrin et al. [3], the individuals who define 
the majority of infectious disease mortality, 
namely neonates and infants of rural 
communities in low-income countries, do so 
in fact because they fall beneath the research 
and health services radar. 
Plant Overview 
A pantropical plant of the Moringaceae 
family, Moringa oleifera, is one of 
approximately thirteen species in the 
monogeneric family [4] [5] [6] [7]. Native to 
the sub-Himalayas of India, M. oleifera has 
been naturalized in various tropical and sub-tropical 
regions of the world, including the 
Middle East, Africa, the Americas, Asia, the 
Philippines, Cambodia, and the Caribbean 
islands [4] [5] [6] [7]. A wide range of 
common names for the tree are documented, 
including benzolive tree, drumstick tree, 
horse-radish tree, kelor tree, mother’s best 
friend, never die tree, mlonge, moonga, 
mulangay and numerous others [5] [6] [7]. 
Due to its tolerance of drought and nutrient 
deficient soils, the perennial softwood is a 
tree with minimal growth needs [6]. As 
such, it can withstand climate conditions 
that range from the high humidity found in 
the tropics to the arid lands of sub-Saharan 
Africa, parts of Asia, and the Middle East 
[4] [5]. Physically, it reaches a maximum 
height of 7-12 meters and a diameter of 20- 
40 cm at roughly 2 meters of height [6]. 
Nutritive Properties 
Asserting its multi-faceted value, the plant is 
utilized for its highly nutritive, medicinal, 
and water purification properties [4]. The 
plant’s nutritive properties are ubiquitous 
throughout the plant, resulting in the 
observation that most plant parts attain 
nutrition and can be eaten: leaves, seeds, 
bark, roots, exudates, flowers, and pods [8]. 
After discovering the plant’s edible nature, 
organizations such as Trees for Life, Church 
World Service, and Educational Concerns 
for Hunger Organization enacted its 
widespread use as a nutritive supplement for 
the malnourished and underserved 
populations of the tropics and subtropics [8]. 
Regarding human micronutrient and 
macronutrient needs, M. oleifera 
quantitatively provides more nutrients per 
gram of plant material than many other plant 
species. For example, gram-for-gram 
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comparisons of M. oleifera leaves (fresh and 
dried) and other common nutritional plant 
sources reveals that M. oleifera provides 
more than seven times the vitamin C found 
in oranges, 10 times the vitamin A found in 
carrots, 17 times the calcium found in milk, 
nine times the protein found in yogurt, 15 
times the potassium found in bananas and 25 
times the iron found in spinach [9] [10]. The 
plant also has high concentrations of 
phosphorus, copper, α-tocopherol, 
riboflavin, nicotinic acid, folic acid, 
pyridoxine, and β-carotene [11]. 
Furthermore, the plant’s leaf structure 
contains significant quantities of the 10 
essential amino acids [9] [10]. 
Water Purification Properties 
M. oleifera has been shown to contain 
water-soluble proteins that act as coagulants. 
Numerous studies have demonstrated that 
M. oleifera seed pumice, when added to 
contaminated water supplies, effectively 
precipitates mineral particulate and various 
organics out of solution. The mechanism of 
action that facilitates precipitation is 
attributed to the ability of charged protein 
molecules to bind and flocculate soluble 
particulate matter [6] [12] [13]. 
Medicinal Properties 
Chemical compounds isolated from M. 
oleifera have been shown to contain useful 
pharmacological properties with prospective 
medicinal applications. A list of possible 
medical applications conferred by M. 
oleifera plant parts includes, but is not 
limited to, antihypertensive, anticancer, 
antispasmodic, antitumor, antiulcer, 
cholesterol lowering, diuretic, 
hepatoprotective, and hypoglycemic 
capabilities, as well as treatment of 
infectious skin and mucosal diseases [1] [4] 
[14]. Leaf extracts have been used to treat 
hyperthyroidism and currently have 
application as an anti-Herpes Simplex Virus 
Type-1 medicine [15] [16]. 
Phytochemical Constituents 
Numerous antibacterial compounds have 
been isolated from M. oleifera, including: 
glucosinolates, rhamanose, pterygospermin, 
and isothiocyanates. Specifically, these 
compounds include 4-(4'-O-acetyl-a-L-rhamnopyranosyloxy) 
benzyl isothiocyanate 
(1), 4-(a-L-rhamnopyranosyloxy)benzyl 
isothiocyanate (2), niazimicin (3), 
pterygospermin (4), benzyl isothiocyanate 
(5), and 4-(a-L-rhamnopyranosyloxy)benzyl 
glucosinolate (6) [8]. 
Since extensive and scientifically rigorous 
studies pertaining to the antibacterial 
activity of M. oleifera seeds began, a 
number of biochemical agents have been 
purported as the component responsible for 
the observed bacterial inhibition. During the 
1940s and 1950s, prior to isolation and 
identification of a specific antibacterial 
agent from the seeds, it was hypothesized 
that pterygospermin was responsible for the 
seeds’ antibacterial activity [8]. 
Pterygospermin, however, was later found to 
dissociate into two benzyl-isothiocyantes, 
compounds known to possess antimicrobial 
properties [8]. Current literature cites the 
isothiocyanate structure and its precursor, 
glucosinolate, as primary constituents from 
M. oleifera seed extracts that confer 
antibacterial activity [17]. 
Research 
It is common practice for researchers to 
scientifically validate the efficacy of 
traditional medicine. It is less common for 
researchers to scientifically validate efforts 
to employ a simple, reproducible means of 
conferring the therapeutic benefits of an 
agent with pre-existing evidence that 
substantiates its putative health benefits. 
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Martin Price, executive director of 
Educational Concerns for Hunger 
Organization (ECHO), published an article 
calling for the development of an antibiotic 
ointment from the seeds of Moringa oleifera, 
noting that the research would be an 
undertaking of how the poor could benefit 
from science [18]. 
The purpose of this study was to investigate 
pragmatic extraction techniques for seed and 
leaf extracts of Moringa oleifera, a plant 
species for which numerous studies have 
demonstrated its antimicrobial efficacy [4] 
[5] [7]. It is hypothesized that bacterial 
inhibition would be conferred – to varying 
degrees – by both plant parts being studied. 
Considering the populations for which this 
research was aimed to benefit (those that 
occupy resource-poor, low income, and 
remote regions of the developing world), the 
phrase ‘pragmatic technique’ is used in this 
paper to define methods that do not require 
sophisticated laboratory equipment (i.e. 
rotary evaporators, gas-/high performance 
liquid chromatography, etc.). Specifically, 
M. oleifera seeds and leaves were extracted 
using: three different solvents – de-ionized 
water (DI H20), inorganic ethanol (95% 
EtOH), and organic ethyl acetate (EA); and 
two different extraction methods – crude and 
sophisticated. Sensitivity disks impregnated 
with the various extracts were used for 
antibiotic susceptibility testing of fourteen 
different bacterial organisms: seven 
representative Gram-negative (G-) species 
and seven representative Gram-positive 
(G+) species. 
METHODS 
I. Plant material preparation 
Leaf powder and whole seeds were obtained 
from Educational Concerns for Hunger 
Organization (ECHO), located in Ft. Myers, 
Florida, U.S.A. Prior to extraction, seeds 
were pulverized via two methods: crude and 
sophisticated. The crude method, chosen for 
its ease in reproducibility and designed for 
use in the most unindustrialized regions of 
the developing world, utilized the crushing 
and grinding of the substrate with a mortar 
and pestle. Alternatively, the sophisticated 
method was propagated by means of an 
ordinary coffee bean grinder. Both methods 
were used until further agitation no longer 
decreased the size gradation of ground plant 
matter. 
II. Antimicrobial constituent extraction 
The extraction process included three 
components. The first consisted of plant 
part: leaf powder (obtained from Moringa 
Farms, California, U.S.A.), and seed 
powder/grinds (obtained from ECHO). The 
second dimension included solvent 
variability. Three different liquid media 
were used to facilitate the extraction process 
of antimicrobial constituents from leaf and 
seed materials: 1) DI H2O, 2) 95% ethanol 
(EtOH), and 3) ethyl acetate (EA). The final 
dimension was created by the previously 
stated dual extraction techniques: crude and 
sophisticated. 
For each extraction, 10 grams of raw plant 
material was macerated with 40 mLs of 
solvent; solvents were heated to boiling 
temperatures and then introduced to small 
containers (either sterilized glassware or tin 
cans depending on the extraction method) 
that contained the plant material. The plant-solvent 
mixture was agitated for 30 s to 
ensure thorough mixture of the components; 
the solution was then covered and allowed 
to steep with no heat stimulus for 15 mins. 
After 15 mins of steeping, the containers 
were each weighed to determine solvent loss 
from evaporation. As needed, excess solvent 
was added to the beakers to normalize the 
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concentrations to 10 grams of plant material 
(PM) per 40 mLs of solvent (crude 
concentration equaling: 250 mg/mL). 
The extraction process (heating solvent, then 
transferring solvent to PM, then macerating 
PM) for the crude technique was performed 
using empty tin cans that were sterilized 
with boiling water within the can and then 
decanting the water. Alternatively, sterile 
(autoclaved) laboratory glass beakers were 
used to facilitate the sophisticated technique. 
After normalization of the plant, liquid 
extract was separated from solid PM by 
filtration using coffee filters. In light of the 
possibility of other modalities, this method 
was chosen because it was economical, 
effective, and extendable to the geographical 
areas of this study’s interest. 
III. Extract impregnation of sensitivity 
disks 
Sterile sensitivity disks were crafted by 
autoclaving 6.5 mm circular disks punched 
from Whatman GF/D Glass Microfibre filter 
disks, using a standard 6.5 mm hole-punch. 
Sterile forceps were used to individually 
submerge sensitivity disks in extracts 
(experimental trials) or solvents (negative 
controls), thus impregnating them. After 
submersion, the disks were flicked to 
remove excess fluid and then placed upon 
aluminum foil plates. These plates were 
heated at 80°C for 45 minutes to dry the 
disks and evaporate residual solvent. 
IV. Organism growth 
Fourteen bacterial organisms were grown at 
37.5°C to assist in determining plant 
constituent efficacy as an antimicrobial 
agent (Table 1). 
Table 1. Three organisms (M. smegmatis, A. faecalis and S. aureus) exhibited susceptibility to 
M. oleifera seed extracts, while one organism (B. sphaericus) demonstrated susceptibility to both 
M. oleifera seed and leaf extracts. 
Organism Biological Classification 
Inhibition 
Seed 
Extracts 
Leaf 
Extracts 
Gram 
Stain Morphology Yes No Yes No 
1 Bacillus sphaericus G+ Bacillus X X 
2 Bacillus subtilis G+ Bacillus X X 
3 Bacillus megaterium G+ Bacillus X X 
4 Bacillus ceresu G+ Bacillus X X 
5 Mycobacterium smegmatis G+ Bacillus X X 
6 Micrococcus luteus G+ Coccus X X 
7 Staphylococcus aureus G+ Coccus X X 
8 Alcaligenes faecalis G- Bacillus X X 
9 Enterobacter aerogenes G- Bacillus X X 
10 Escherichia coli G- Bacillus X X 
11 Klebsiella pneumoniae G- Bacillus X X 
12 Proteus mirabilis G- Bacillus X X 
13 Proteus vulgaris G- Bacillus X X 
14 Shigella flexneri G- Bacillus X X 
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Organisms were attained from American 
Type Culture Collection. Agar slants, 
incubated at 37.5°C were used as perpetual 
growth sites for the organisms. Seven days 
before plating, the organisms were 
transferred to tryptic soy broth (TSB), the 
medium in which they remained until 
susceptibility testing was performed. 
Transference of 75 μLs of TSB (containing 
the respective microorganism) to sterile LB 
agar plates was followed with a glass 
hockey-stick method of evenly distributing 
the organism across the plate and ensuring 
confluent bacterial growth. 
Five sets of organisms were prepared to 
correlate with extract methods and plant part 
(4); a final set of organisms were prepared to 
examine positive and negative controls. 
Three agar plate replicates were prepared for 
each organism within all 5 sets. 
V. Disk implantation 
Immediately following dissemination of 
bacterial TSB onto LB agar plates, 
sensitivity disks were implanted onto the 
plate. Three sensitivity disks, each 
correlating to a specified extract solvent, 
were placed on each replicate within the 
four extraction sets. Six sensitivity disks, 
which correlated to the positive 
(Vancomycin, Penicillin, and Tetracycline) 
and negative (pure solvent without extract) 
controls, were placed on each replicate 
within the control set. A rectangular shape 
beneath the agar plate was used for 
standardized orientation of the plate and 
subsequent placement of each disk. 
VI. Incubation and reading of dishes 
Agar plates were incubated at 37.5°C for a 
total duration of 72 hrs, and zones of 
inhibition (mm) were recorded at 24, 48, and 
72 hrs post disk-placement. 
RESULTS 
I. Plant part and solvent 
Leaf and seed extracts prepared with 95% 
EtOH or EA conferred no inhibition among 
the 14 organisms. Extracts prepared using 
M. oleifera leaves with DI H2O exhibited 
antibacterial inhibition against one of the 
fourteen bacteria: Bacillus sphaericus (Table 
1). M. oleifera seed extracts prepared with 
DI H2O conferred inhibition among four of 
the 14 bacteria: B. sphaericus, 
Mycobacterium smegmatis, Staphylococcus 
aureus, and Alcaligenes faecalis (Table 1). 
II. Susceptible species 
A total of four bacterial species were 
susceptible to inhibition by either leaf or 
seed extracts prepared from M. oleifera 
(Table 1). One species, B. sphaericus, 
showed susceptibility to both seed and leaf 
extracts. M. smegmatis, S. aureus, and A. 
faecalis were susceptible to seed extracts but 
were resistant to leaf extracts. Of the 
species susceptible to seed extracts, bacterial 
inhibition was phylogenetically and 
morphologically scattered; the extracts were 
effective against representative species of 
G+ bacillus (B. sphaericus and M. 
smegmatis), G+ coccus (S. aureus), and G-bacillus 
(A. faecalis). One common trend 
was the indication that 75% of the 
organisms inhibited by seed extracts were 
G+ while the remaining 25% were G-; 
further 75% were bacilli species, whereas 
the remaining 25% were cocci species. 
III. Method variation 
(Results are displayed as Average ± 
Standard Error) 
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Each extraction for the given plant part and 
solvent was performed using two extraction 
methods: crude, and sophisticated. The leaf-water 
extract prepared using sophisticated 
methods demonstrated antibacterial efficacy 
against B. sphaericus (4.11mm ± 0.11), 
while the leaf-water extract prepared using 
crude methods did not inhibit the organism. 
Of the four organisms susceptible to seed-water 
extracts, two were susceptible to both 
crude and sophisticated extraction methods, 
while the remaining two were susceptible to 
only one method (Table 2). 
Table 2. Utilizing sophisticated and crude methods, DI H2O M. oleifera seed extracts 
demonstrated statistically significant zones of inhibition and provided statistically similar results 
to penicillin and/or tetracycline with regard to four pathogens. 
Organisms Inhibited 
by Seed DI H2O 
Extract 
Bacterial Inhibition Method Observed 
Antibiotic 
Efficacy 
Antibiotics for Which Seed 
Extracts Demonstrated 
Statistically Comparable 
Results 
Sophisticated Crude V P T 
1 Bacillus sphaericus X X V, P & T X 
2 Mycobacterium 
smegmatis X T X 
3 Staphylococcus 
aureus X T X X 
4 Alcaligenes faecalis X X V, P & T X 
(Where: V = vancomycin, P = penicillin, and T = tetracycline) 
B. sphaericus and A. faecalis demonstrated 
susceptibility to both extraction methods for 
the extracts. The sophisticated method 
produced a wider zone of inhibition 
(diameter) for B.sphaericus, crude (6.44mm 
± 1.97) and sophisticated (11.22mm ± 0.29); 
the crude method, however, demonstrated an 
increased zone of inhibition (diameter) for 
A. faecalis (crude = 5.67mm ± 1.92 and 
sophisticated = 1.67mm ± 0.19). M. 
smegmatis was inhibited by crude extract 
(2.0mm ± 1.73), but showed no resistance to 
the sophisticated extract. S. aureus was 
susceptible to sophisticated extracts 
(1.78mm ± 0.91), but not crude seed-water 
extracts. For all organisms, no significant 
difference in zone of inhibition was 
observed between the two extraction 
methods (P >0.1 or P > 0.2). 
IV. Controls versus extracts 
Each organism examined was subjected to 
susceptibility tests with negative and 
positive controls (NCs and PCs). 
Susceptibility to NCs, PCs, and plant part 
extracts were compared using zone of 
inhibition (ZOI) measurements. NCs 
consisted of sensitivity disks soaked in pure 
solvent: DI H2O, 95% EtOH, or EA. None 
of the NCs were found to confer inhibition 
among the fourteen organisms. 
PCs consisted of three known antibiotic 
agents: penicillin, tetracycline, and 
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vancomycin. Species susceptible to both 
penicillin and the seed extracts, namely B. 
sphaericus and A. faecalis, did not 
demonstrate a significant difference between 
the average ZOI produced by penicillin or 
the seed extracts. Similar to penicillin, 
species susceptible to tetracycline and seed 
extracts, with the exception of B. sphaericus, 
demonstrated comparable average ZOIs, that 
is, ZOIs were not significantly different 
between tetracycline disks and seed extract 
disks. Alternatively, for organisms 
susceptible to vancomycin and seed extracts 
– B. sphaericus and A. faecalis – 
vancomycin disks resulted in significantly 
elevated ZOIs relative to the seed extracts. 
DISCUSSION 
I. Plant Part and Solvent 
In discussing the general inhibitory efficacy 
of leaf and seed extracts from this research, 
regrowth must be considered. Regrowth is a 
biological phenomenon whereby organismal 
reproduction/growth is inhibited for a short 
duration, but is subsequently followed by 
proliferation of the organism. Because data 
collections were performed at 24-hr 
intervals, regrowth of organisms prior to the 
first data collection (24-hrs) or between each 
of the 24-hr intervals must not be ruled out 
and may account for the lack of observed 
organismal inhibition by leaf or seed 
extracts. 
Plant Part 
Of the fourteen organisms examined, seed 
extracts were more effective antibacterial 
inhibitors than leaf extracts. In fact, several 
trials exhibited microbial proliferation near 
and/or on the sensitivity disks soaked in leaf 
extract. It is suspected that proliferation was 
due to the nutrient dense structure of M. 
oleifera leaves [8]. Similarly, for organisms 
that were not inhibited by seed extracts, 
proliferation of bacterial colonies was also 
observed near and/or on the sensitivity disks 
soaked in seed extract. Again, this may also 
be attributed to the nutrient rich nature of the 
plant’s seeds [8]. Thus, given the abundance 
of vitamins, minerals, and amino acids 
found within the leaves [4] [5] [7] [19], 
coupled with the populations this research 
aimed to address, it can be reasonably 
concluded that leaf material would be more 
appropriately allocated for use as a 
nutritional supplement rather than a source 
of antibacterial components. 
Solvent 
The polarity of water is markedly elevated 
relative to EtOH and EA. It is possible that 
variations in solvent polarity, as measured 
by dielectric constants, explains why EtOH 
and EA extracts did not attract, bind, and 
thus extract charged antimicrobial 
constituents. Further, it is also possible that 
the 95% EtOH extract used, may have 
degraded proteins implicated in inhibitory 
properties, thus debilitating the extract’s 
ability to confer bacterial inhibition. 
II. Method Variation 
The primary objective underlying the 
experimental design for this study was the 
determination as to whether crude extraction 
methods were capable of inhibiting bacterial 
growth. The absence of statistical evidence 
pointing toward significant differences 
between ZOIs conferred by crude and 
sophisticated extraction methods implicates 
that for certain bacterial species, the crude 
method of extraction is just as effective as 
the sophisticated method. These findings 
support the prospect of crude extraction as a 
pragmatic method of extracting antibacterial 
agents from M. oleifera in resource-poor 
settings. 
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III. Susceptible Species 
Despite phylogenetic diversity among 
species susceptible to M. oleifera extracts, 
results demonstrated a common trend in that 
M. oleifera seed extracts were three times 
more effective against G+ species compared 
to G- species based upon the species 
observed. It is suspected that these 
differences were observed due to variation 
between the cell wall composition of G+ and 
G- bacteria. Perhaps the second 
phospholipid membrane bilayer of G-bacteria 
helped these species evade 
antibiotic degradation from the peptides 
within the seed extract [20]. 
IV. Controls versus Extract 
The lack of significant difference between 
ZOIs produced by M. oleifera seed extracts 
compared to those produced by penicillin 
and tetracycline implicate comparable 
antibacterial efficacy in susceptible taxa 
between M. oleifera seed extracts and the 
aforementioned antibiotics. These results 
encouraged the researchers of this study to 
examine the biochemical mechanism of 
action for penicillin and tetracycline, and 
then use such activity to hypothesize 
possible mechanisms of action yielded by 
seed extracts. 
Administered at clinical doses, antibiotics 
that inhibit the proliferation of bacteria are 
classified as either: bactericidal or 
bacteriostatic. The former prevent bacterial 
proliferation by killing the organism, 
whereas the latter prevents bacterial 
proliferation by preventing the organism 
from dividing, which in turn assists the 
individual’s immune system to destroy the 
pathogen. Penicillin is a bacteriostatic β- 
lactam antibiotic derived from a fungi 
source, and is effective in destroying G+ 
bacteria. Its mechanism of action is such that 
it inhibits bacterial cell wall formation by 
blocking the cross-linking of structures 
within the cell wall (i.e. cell wall synthesis) 
[20]. Tetracyline, however, is a broad-spectrum 
bacteriostatic antibiotic, 
functioning as a G+ and G- antibiotic. 
Unlike penicillin, tetracycline inhibits 
bacterial growth intracellularly by binding to 
the bacterial 30S ribosomal subunit, thus 
disrupting protein synthesis [20]. 
The mechanism by which M. oleifera 
derived isothiocyanates result in bacterial 
inhibition is not understood to date. 
However, extensive research documenting 
the efficacy of synthetic, highly active 
antibacterial peptides derived from M. 
oleifera seed proteins has helped elucidate 
the mechanism by which the cationic 
peptide results in microbial inhibition [13]. 
The secondary structure of the antimicrobial 
polypeptide contains positively charged α- 
helices that are thought to bind to negatively 
charged phospholipid heads of the bacterial 
cell membrane; the charge attraction and 
stabilization allows for subsequent 
interaction between a loop region in the 
antibacterial peptide structure and aliphatic 
fatty acids of the bacterial membrane [13]. 
In turn, the bacterial membrane is 
destabilized by changes in permeability and 
lipid distribution, as well as disruption of its 
membrane potential. In sum, these effects 
result in breaks in the bacterial cell 
membrane, disaggregation of its 
components, and eventually cell death [12] 
[13]. 
With this understanding, antibiotic peptides 
of M. oleifera seed extracts were expected to 
exhibit inhibition comparable to that of 
penicillin; however, they were not expected 
to produce results similar to tetracycline. 
Given that tetracycline works intracellularly 
as a bacteriostatic antibiotic, it is possible 
that other constituents of M. oleifera seed 
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extracts, such as the identified 
isothiocyanates, also work intracellularly in 
a concerted effort with the antibiotic 
peptides. 
V. Future Research 
In futures trials it would be desirable to 
repeat the leaf and seed DI H2O extract 
experimentation, withholding the 80°C 
incubation that evaporated the solvent. It is 
suspected that motility afforded to microbial 
inhibiting molecules by the presence of DI 
H2O will allow the molecules to disperse 
across the agar lawn with greater 
confluence. Repeated experiments could 
also rule out the possibility of regrowth by 
collecting data samples over durations 
shorter than 24-hrs, i.e. 3-hr, 6-hr, 9-hr, or 
12-hr time intervals. 
Although several of the organisms tested in 
this study are known to be common 
colonizers of human skin and 
gastrointestinal tracts, it is recommended 
that antibacterial susceptibility tests, using 
M. oleifera extracts, be performed against 
pathogenic bacteria endemic to resource-poor 
settings. Further, it is also suggested 
that MIC studies be conducted using the 
crude extraction technique. If the extracts 
prove to be efficacious inhibitors of 
pathogenic bacteria, then shelf-life studies 
should be performed to further qualify this 
pragmatic extraction technique that it may 
be used in destitute regions worldwide. 
CONCLUSIONS 
This study demonstrated that when M. 
oleifera seeds and leaves are prepared using 
very crude methods they can provide, to 
varying abilities, antimicrobial capabilities 
comparable to some contemporary remedies 
against common pathogens that cause 
human morbidity. This research is a 
reminder that heroic lengths and modern 
science are not always necessary to combat 
antimicrobial pathogens in remote regions 
where modern medicine is not available. 
ACKNOWLEDGEMENTS 
Many thanks to Educational Concerns for 
Hunger Organization (ECHO), located in Ft. 
Myers, Florida, U.S.A. and Moringa Farms, 
located in Sherman Oaks, California, U.S.A. 
for their abundant guidance and provision of 
plant part specimens for this and future 
research endeavors. 
REFERENCES 
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Potential uses-of-moringa-oleifera

  • 1. INTERNATIONAL JOURNAL OF PHYTOTHEARPY RESEARCH ISSN 2278 – 5701 Original Research Article POTENTIAL USES OF Moringa oleifera AND AN EXAMINATION OF ANTIBIOTIC EFFICACY CONFERRED BY M. oleifera SEED AND LEAF EXTRACTS USING CRUDE EXTRACTION TECHNIQUES AVAILABLE TO UNDERSERVED INDIGENOUS POPULATIONS Rockwood, J.L.1*,Anderson, B.G.2*,Casamatta, D.A.3 1 MS4, The University of Texas Health Science Center, San Antonio, TX, USA 2 DO, Lake Erie College of Osteopathic Medicine, Erie, PA, USA 3 PhD, University of North Florida, Jacksonville, FL, USA ABSTRACT: Moringa oleifera, a pantropical plant, is one of approximately thirteen species belonging to the monogeneric Moringaceae family. Ethnobotanical studies conducted in Guatemala found that one of the primary medicinal purposes of M. oleifera was its use for the treatment of infectious skin and mucosal diseases. As it is common practice for researchers to scientifically validate the efficacy of traditional medicine, it is less common for researchers to scientifically validate simple, reproducible means of conferring therapeutic benefits of plant parts. This study was conducted to investigate pragmatic extraction techniques for seed and leaf extracts of M. oleifera, a plant species for which numerous studies have demonstrated its antimicrobial efficacy. M. oleifera seeds and leaves were extracted using three different solvents (de-ionized water, inorganic ethanol, organic ethyl acetate) and two different extraction methods (crude, sophisticated). Sensitivity disks impregnated with the various extracts were used for antibiotic susceptibility testing of fourteen bacterial species: seven representative Gram-negative and seven representative Gram-positive. De-ionized water was the only solvent capable of extracting plant constituents which conferred bacterial inhibition. Seed extracts were found to inhibit a broader range of organisms (4) than leaf extracts (1). 75% of the organisms inhibited by seed extracts were Gram-positive bacteria. A single parameter, the zone of inhibition, was used to compare antibacterial efficacy between extraction methods, trials, and controls. No difference was observed between the zone of inhibition of crude and sophisticated extracts. Seed extracts demonstrated a zone of inhibition comparable to that of penicillin and tetracycline. Keywords:Moringa oleifera,Crude extraction,Phytomedicine 61 www.earthjournals.org Volume 3 Issue 2 2013
  • 2. INTERNATIONAL JOURNAL OF PHYTOTHEARPY RESEARCH ISSN 2278 – 5701 INTRODUCTION: Antibiotic drugs are one of the many tools in our current arsenal of medical defense. Penicillin is a prime example of how modern antibiotics revolutionized our capacity to combat morbidity and mortality resulting from bacterial infections. Despite great strides and advances in medical equipment and therapies, infectious diseases have long remained the leading cause of morbidity and mortality in the developing world [1]. A sober approach to examining the underlying cause for such disparities requires acknowledging that within the current climate of market-driven economies and for-profit pharmaceutical practices, people often suffer from curable and preventable diseases due to the expenses associated with research, development, and delivery of healthcare [2]. Resources elucidating current research efforts and statistical data for the ‘big-three’ communicable diseases – malaria (resulting from infection with Plasmodium species), AIDS (resulting from infection with HIV), and tuberculosis (resulting from infection with Mycobacterium species) - abound the annals of scientific literature. Nevertheless, documentation of research aimed toward pragmatic and reproducible antibiotic therapies for residents of resource-poor settings is comparatively small. According to Osrin et al. [3], the individuals who define the majority of infectious disease mortality, namely neonates and infants of rural communities in low-income countries, do so in fact because they fall beneath the research and health services radar. Plant Overview A pantropical plant of the Moringaceae family, Moringa oleifera, is one of approximately thirteen species in the monogeneric family [4] [5] [6] [7]. Native to the sub-Himalayas of India, M. oleifera has been naturalized in various tropical and sub-tropical regions of the world, including the Middle East, Africa, the Americas, Asia, the Philippines, Cambodia, and the Caribbean islands [4] [5] [6] [7]. A wide range of common names for the tree are documented, including benzolive tree, drumstick tree, horse-radish tree, kelor tree, mother’s best friend, never die tree, mlonge, moonga, mulangay and numerous others [5] [6] [7]. Due to its tolerance of drought and nutrient deficient soils, the perennial softwood is a tree with minimal growth needs [6]. As such, it can withstand climate conditions that range from the high humidity found in the tropics to the arid lands of sub-Saharan Africa, parts of Asia, and the Middle East [4] [5]. Physically, it reaches a maximum height of 7-12 meters and a diameter of 20- 40 cm at roughly 2 meters of height [6]. Nutritive Properties Asserting its multi-faceted value, the plant is utilized for its highly nutritive, medicinal, and water purification properties [4]. The plant’s nutritive properties are ubiquitous throughout the plant, resulting in the observation that most plant parts attain nutrition and can be eaten: leaves, seeds, bark, roots, exudates, flowers, and pods [8]. After discovering the plant’s edible nature, organizations such as Trees for Life, Church World Service, and Educational Concerns for Hunger Organization enacted its widespread use as a nutritive supplement for the malnourished and underserved populations of the tropics and subtropics [8]. Regarding human micronutrient and macronutrient needs, M. oleifera quantitatively provides more nutrients per gram of plant material than many other plant species. For example, gram-for-gram 62 www.earthjournals.org Volume 3 Issue 2 2013
  • 3. INTERNATIONAL JOURNAL OF PHYTOTHEARPY RESEARCH ISSN 2278 – 5701 comparisons of M. oleifera leaves (fresh and dried) and other common nutritional plant sources reveals that M. oleifera provides more than seven times the vitamin C found in oranges, 10 times the vitamin A found in carrots, 17 times the calcium found in milk, nine times the protein found in yogurt, 15 times the potassium found in bananas and 25 times the iron found in spinach [9] [10]. The plant also has high concentrations of phosphorus, copper, α-tocopherol, riboflavin, nicotinic acid, folic acid, pyridoxine, and β-carotene [11]. Furthermore, the plant’s leaf structure contains significant quantities of the 10 essential amino acids [9] [10]. Water Purification Properties M. oleifera has been shown to contain water-soluble proteins that act as coagulants. Numerous studies have demonstrated that M. oleifera seed pumice, when added to contaminated water supplies, effectively precipitates mineral particulate and various organics out of solution. The mechanism of action that facilitates precipitation is attributed to the ability of charged protein molecules to bind and flocculate soluble particulate matter [6] [12] [13]. Medicinal Properties Chemical compounds isolated from M. oleifera have been shown to contain useful pharmacological properties with prospective medicinal applications. A list of possible medical applications conferred by M. oleifera plant parts includes, but is not limited to, antihypertensive, anticancer, antispasmodic, antitumor, antiulcer, cholesterol lowering, diuretic, hepatoprotective, and hypoglycemic capabilities, as well as treatment of infectious skin and mucosal diseases [1] [4] [14]. Leaf extracts have been used to treat hyperthyroidism and currently have application as an anti-Herpes Simplex Virus Type-1 medicine [15] [16]. Phytochemical Constituents Numerous antibacterial compounds have been isolated from M. oleifera, including: glucosinolates, rhamanose, pterygospermin, and isothiocyanates. Specifically, these compounds include 4-(4'-O-acetyl-a-L-rhamnopyranosyloxy) benzyl isothiocyanate (1), 4-(a-L-rhamnopyranosyloxy)benzyl isothiocyanate (2), niazimicin (3), pterygospermin (4), benzyl isothiocyanate (5), and 4-(a-L-rhamnopyranosyloxy)benzyl glucosinolate (6) [8]. Since extensive and scientifically rigorous studies pertaining to the antibacterial activity of M. oleifera seeds began, a number of biochemical agents have been purported as the component responsible for the observed bacterial inhibition. During the 1940s and 1950s, prior to isolation and identification of a specific antibacterial agent from the seeds, it was hypothesized that pterygospermin was responsible for the seeds’ antibacterial activity [8]. Pterygospermin, however, was later found to dissociate into two benzyl-isothiocyantes, compounds known to possess antimicrobial properties [8]. Current literature cites the isothiocyanate structure and its precursor, glucosinolate, as primary constituents from M. oleifera seed extracts that confer antibacterial activity [17]. Research It is common practice for researchers to scientifically validate the efficacy of traditional medicine. It is less common for researchers to scientifically validate efforts to employ a simple, reproducible means of conferring the therapeutic benefits of an agent with pre-existing evidence that substantiates its putative health benefits. 63 www.earthjournals.org Volume 3 Issue 2 2013
  • 4. INTERNATIONAL JOURNAL OF PHYTOTHEARPY RESEARCH ISSN 2278 – 5701 Martin Price, executive director of Educational Concerns for Hunger Organization (ECHO), published an article calling for the development of an antibiotic ointment from the seeds of Moringa oleifera, noting that the research would be an undertaking of how the poor could benefit from science [18]. The purpose of this study was to investigate pragmatic extraction techniques for seed and leaf extracts of Moringa oleifera, a plant species for which numerous studies have demonstrated its antimicrobial efficacy [4] [5] [7]. It is hypothesized that bacterial inhibition would be conferred – to varying degrees – by both plant parts being studied. Considering the populations for which this research was aimed to benefit (those that occupy resource-poor, low income, and remote regions of the developing world), the phrase ‘pragmatic technique’ is used in this paper to define methods that do not require sophisticated laboratory equipment (i.e. rotary evaporators, gas-/high performance liquid chromatography, etc.). Specifically, M. oleifera seeds and leaves were extracted using: three different solvents – de-ionized water (DI H20), inorganic ethanol (95% EtOH), and organic ethyl acetate (EA); and two different extraction methods – crude and sophisticated. Sensitivity disks impregnated with the various extracts were used for antibiotic susceptibility testing of fourteen different bacterial organisms: seven representative Gram-negative (G-) species and seven representative Gram-positive (G+) species. METHODS I. Plant material preparation Leaf powder and whole seeds were obtained from Educational Concerns for Hunger Organization (ECHO), located in Ft. Myers, Florida, U.S.A. Prior to extraction, seeds were pulverized via two methods: crude and sophisticated. The crude method, chosen for its ease in reproducibility and designed for use in the most unindustrialized regions of the developing world, utilized the crushing and grinding of the substrate with a mortar and pestle. Alternatively, the sophisticated method was propagated by means of an ordinary coffee bean grinder. Both methods were used until further agitation no longer decreased the size gradation of ground plant matter. II. Antimicrobial constituent extraction The extraction process included three components. The first consisted of plant part: leaf powder (obtained from Moringa Farms, California, U.S.A.), and seed powder/grinds (obtained from ECHO). The second dimension included solvent variability. Three different liquid media were used to facilitate the extraction process of antimicrobial constituents from leaf and seed materials: 1) DI H2O, 2) 95% ethanol (EtOH), and 3) ethyl acetate (EA). The final dimension was created by the previously stated dual extraction techniques: crude and sophisticated. For each extraction, 10 grams of raw plant material was macerated with 40 mLs of solvent; solvents were heated to boiling temperatures and then introduced to small containers (either sterilized glassware or tin cans depending on the extraction method) that contained the plant material. The plant-solvent mixture was agitated for 30 s to ensure thorough mixture of the components; the solution was then covered and allowed to steep with no heat stimulus for 15 mins. After 15 mins of steeping, the containers were each weighed to determine solvent loss from evaporation. As needed, excess solvent was added to the beakers to normalize the 64 www.earthjournals.org Volume 3 Issue 2 2013
  • 5. INTERNATIONAL JOURNAL OF PHYTOTHEARPY RESEARCH ISSN 2278 – 5701 concentrations to 10 grams of plant material (PM) per 40 mLs of solvent (crude concentration equaling: 250 mg/mL). The extraction process (heating solvent, then transferring solvent to PM, then macerating PM) for the crude technique was performed using empty tin cans that were sterilized with boiling water within the can and then decanting the water. Alternatively, sterile (autoclaved) laboratory glass beakers were used to facilitate the sophisticated technique. After normalization of the plant, liquid extract was separated from solid PM by filtration using coffee filters. In light of the possibility of other modalities, this method was chosen because it was economical, effective, and extendable to the geographical areas of this study’s interest. III. Extract impregnation of sensitivity disks Sterile sensitivity disks were crafted by autoclaving 6.5 mm circular disks punched from Whatman GF/D Glass Microfibre filter disks, using a standard 6.5 mm hole-punch. Sterile forceps were used to individually submerge sensitivity disks in extracts (experimental trials) or solvents (negative controls), thus impregnating them. After submersion, the disks were flicked to remove excess fluid and then placed upon aluminum foil plates. These plates were heated at 80°C for 45 minutes to dry the disks and evaporate residual solvent. IV. Organism growth Fourteen bacterial organisms were grown at 37.5°C to assist in determining plant constituent efficacy as an antimicrobial agent (Table 1). Table 1. Three organisms (M. smegmatis, A. faecalis and S. aureus) exhibited susceptibility to M. oleifera seed extracts, while one organism (B. sphaericus) demonstrated susceptibility to both M. oleifera seed and leaf extracts. Organism Biological Classification Inhibition Seed Extracts Leaf Extracts Gram Stain Morphology Yes No Yes No 1 Bacillus sphaericus G+ Bacillus X X 2 Bacillus subtilis G+ Bacillus X X 3 Bacillus megaterium G+ Bacillus X X 4 Bacillus ceresu G+ Bacillus X X 5 Mycobacterium smegmatis G+ Bacillus X X 6 Micrococcus luteus G+ Coccus X X 7 Staphylococcus aureus G+ Coccus X X 8 Alcaligenes faecalis G- Bacillus X X 9 Enterobacter aerogenes G- Bacillus X X 10 Escherichia coli G- Bacillus X X 11 Klebsiella pneumoniae G- Bacillus X X 12 Proteus mirabilis G- Bacillus X X 13 Proteus vulgaris G- Bacillus X X 14 Shigella flexneri G- Bacillus X X 65 www.earthjournals.org Volume 3 Issue 2 2013
  • 6. INTERNATIONAL JOURNAL OF PHYTOTHEARPY RESEARCH ISSN 2278 – 5701 Organisms were attained from American Type Culture Collection. Agar slants, incubated at 37.5°C were used as perpetual growth sites for the organisms. Seven days before plating, the organisms were transferred to tryptic soy broth (TSB), the medium in which they remained until susceptibility testing was performed. Transference of 75 μLs of TSB (containing the respective microorganism) to sterile LB agar plates was followed with a glass hockey-stick method of evenly distributing the organism across the plate and ensuring confluent bacterial growth. Five sets of organisms were prepared to correlate with extract methods and plant part (4); a final set of organisms were prepared to examine positive and negative controls. Three agar plate replicates were prepared for each organism within all 5 sets. V. Disk implantation Immediately following dissemination of bacterial TSB onto LB agar plates, sensitivity disks were implanted onto the plate. Three sensitivity disks, each correlating to a specified extract solvent, were placed on each replicate within the four extraction sets. Six sensitivity disks, which correlated to the positive (Vancomycin, Penicillin, and Tetracycline) and negative (pure solvent without extract) controls, were placed on each replicate within the control set. A rectangular shape beneath the agar plate was used for standardized orientation of the plate and subsequent placement of each disk. VI. Incubation and reading of dishes Agar plates were incubated at 37.5°C for a total duration of 72 hrs, and zones of inhibition (mm) were recorded at 24, 48, and 72 hrs post disk-placement. RESULTS I. Plant part and solvent Leaf and seed extracts prepared with 95% EtOH or EA conferred no inhibition among the 14 organisms. Extracts prepared using M. oleifera leaves with DI H2O exhibited antibacterial inhibition against one of the fourteen bacteria: Bacillus sphaericus (Table 1). M. oleifera seed extracts prepared with DI H2O conferred inhibition among four of the 14 bacteria: B. sphaericus, Mycobacterium smegmatis, Staphylococcus aureus, and Alcaligenes faecalis (Table 1). II. Susceptible species A total of four bacterial species were susceptible to inhibition by either leaf or seed extracts prepared from M. oleifera (Table 1). One species, B. sphaericus, showed susceptibility to both seed and leaf extracts. M. smegmatis, S. aureus, and A. faecalis were susceptible to seed extracts but were resistant to leaf extracts. Of the species susceptible to seed extracts, bacterial inhibition was phylogenetically and morphologically scattered; the extracts were effective against representative species of G+ bacillus (B. sphaericus and M. smegmatis), G+ coccus (S. aureus), and G-bacillus (A. faecalis). One common trend was the indication that 75% of the organisms inhibited by seed extracts were G+ while the remaining 25% were G-; further 75% were bacilli species, whereas the remaining 25% were cocci species. III. Method variation (Results are displayed as Average ± Standard Error) 66 www.earthjournals.org Volume 3 Issue 2 2013
  • 7. INTERNATIONAL JOURNAL OF PHYTOTHEARPY RESEARCH ISSN 2278 – 5701 Each extraction for the given plant part and solvent was performed using two extraction methods: crude, and sophisticated. The leaf-water extract prepared using sophisticated methods demonstrated antibacterial efficacy against B. sphaericus (4.11mm ± 0.11), while the leaf-water extract prepared using crude methods did not inhibit the organism. Of the four organisms susceptible to seed-water extracts, two were susceptible to both crude and sophisticated extraction methods, while the remaining two were susceptible to only one method (Table 2). Table 2. Utilizing sophisticated and crude methods, DI H2O M. oleifera seed extracts demonstrated statistically significant zones of inhibition and provided statistically similar results to penicillin and/or tetracycline with regard to four pathogens. Organisms Inhibited by Seed DI H2O Extract Bacterial Inhibition Method Observed Antibiotic Efficacy Antibiotics for Which Seed Extracts Demonstrated Statistically Comparable Results Sophisticated Crude V P T 1 Bacillus sphaericus X X V, P & T X 2 Mycobacterium smegmatis X T X 3 Staphylococcus aureus X T X X 4 Alcaligenes faecalis X X V, P & T X (Where: V = vancomycin, P = penicillin, and T = tetracycline) B. sphaericus and A. faecalis demonstrated susceptibility to both extraction methods for the extracts. The sophisticated method produced a wider zone of inhibition (diameter) for B.sphaericus, crude (6.44mm ± 1.97) and sophisticated (11.22mm ± 0.29); the crude method, however, demonstrated an increased zone of inhibition (diameter) for A. faecalis (crude = 5.67mm ± 1.92 and sophisticated = 1.67mm ± 0.19). M. smegmatis was inhibited by crude extract (2.0mm ± 1.73), but showed no resistance to the sophisticated extract. S. aureus was susceptible to sophisticated extracts (1.78mm ± 0.91), but not crude seed-water extracts. For all organisms, no significant difference in zone of inhibition was observed between the two extraction methods (P >0.1 or P > 0.2). IV. Controls versus extracts Each organism examined was subjected to susceptibility tests with negative and positive controls (NCs and PCs). Susceptibility to NCs, PCs, and plant part extracts were compared using zone of inhibition (ZOI) measurements. NCs consisted of sensitivity disks soaked in pure solvent: DI H2O, 95% EtOH, or EA. None of the NCs were found to confer inhibition among the fourteen organisms. PCs consisted of three known antibiotic agents: penicillin, tetracycline, and 67 www.earthjournals.org Volume 3 Issue 2 2013
  • 8. INTERNATIONAL JOURNAL OF PHYTOTHEARPY RESEARCH ISSN 2278 – 5701 vancomycin. Species susceptible to both penicillin and the seed extracts, namely B. sphaericus and A. faecalis, did not demonstrate a significant difference between the average ZOI produced by penicillin or the seed extracts. Similar to penicillin, species susceptible to tetracycline and seed extracts, with the exception of B. sphaericus, demonstrated comparable average ZOIs, that is, ZOIs were not significantly different between tetracycline disks and seed extract disks. Alternatively, for organisms susceptible to vancomycin and seed extracts – B. sphaericus and A. faecalis – vancomycin disks resulted in significantly elevated ZOIs relative to the seed extracts. DISCUSSION I. Plant Part and Solvent In discussing the general inhibitory efficacy of leaf and seed extracts from this research, regrowth must be considered. Regrowth is a biological phenomenon whereby organismal reproduction/growth is inhibited for a short duration, but is subsequently followed by proliferation of the organism. Because data collections were performed at 24-hr intervals, regrowth of organisms prior to the first data collection (24-hrs) or between each of the 24-hr intervals must not be ruled out and may account for the lack of observed organismal inhibition by leaf or seed extracts. Plant Part Of the fourteen organisms examined, seed extracts were more effective antibacterial inhibitors than leaf extracts. In fact, several trials exhibited microbial proliferation near and/or on the sensitivity disks soaked in leaf extract. It is suspected that proliferation was due to the nutrient dense structure of M. oleifera leaves [8]. Similarly, for organisms that were not inhibited by seed extracts, proliferation of bacterial colonies was also observed near and/or on the sensitivity disks soaked in seed extract. Again, this may also be attributed to the nutrient rich nature of the plant’s seeds [8]. Thus, given the abundance of vitamins, minerals, and amino acids found within the leaves [4] [5] [7] [19], coupled with the populations this research aimed to address, it can be reasonably concluded that leaf material would be more appropriately allocated for use as a nutritional supplement rather than a source of antibacterial components. Solvent The polarity of water is markedly elevated relative to EtOH and EA. It is possible that variations in solvent polarity, as measured by dielectric constants, explains why EtOH and EA extracts did not attract, bind, and thus extract charged antimicrobial constituents. Further, it is also possible that the 95% EtOH extract used, may have degraded proteins implicated in inhibitory properties, thus debilitating the extract’s ability to confer bacterial inhibition. II. Method Variation The primary objective underlying the experimental design for this study was the determination as to whether crude extraction methods were capable of inhibiting bacterial growth. The absence of statistical evidence pointing toward significant differences between ZOIs conferred by crude and sophisticated extraction methods implicates that for certain bacterial species, the crude method of extraction is just as effective as the sophisticated method. These findings support the prospect of crude extraction as a pragmatic method of extracting antibacterial agents from M. oleifera in resource-poor settings. 68 www.earthjournals.org Volume 3 Issue 2 2013
  • 9. INTERNATIONAL JOURNAL OF PHYTOTHEARPY RESEARCH ISSN 2278 – 5701 III. Susceptible Species Despite phylogenetic diversity among species susceptible to M. oleifera extracts, results demonstrated a common trend in that M. oleifera seed extracts were three times more effective against G+ species compared to G- species based upon the species observed. It is suspected that these differences were observed due to variation between the cell wall composition of G+ and G- bacteria. Perhaps the second phospholipid membrane bilayer of G-bacteria helped these species evade antibiotic degradation from the peptides within the seed extract [20]. IV. Controls versus Extract The lack of significant difference between ZOIs produced by M. oleifera seed extracts compared to those produced by penicillin and tetracycline implicate comparable antibacterial efficacy in susceptible taxa between M. oleifera seed extracts and the aforementioned antibiotics. These results encouraged the researchers of this study to examine the biochemical mechanism of action for penicillin and tetracycline, and then use such activity to hypothesize possible mechanisms of action yielded by seed extracts. Administered at clinical doses, antibiotics that inhibit the proliferation of bacteria are classified as either: bactericidal or bacteriostatic. The former prevent bacterial proliferation by killing the organism, whereas the latter prevents bacterial proliferation by preventing the organism from dividing, which in turn assists the individual’s immune system to destroy the pathogen. Penicillin is a bacteriostatic β- lactam antibiotic derived from a fungi source, and is effective in destroying G+ bacteria. Its mechanism of action is such that it inhibits bacterial cell wall formation by blocking the cross-linking of structures within the cell wall (i.e. cell wall synthesis) [20]. Tetracyline, however, is a broad-spectrum bacteriostatic antibiotic, functioning as a G+ and G- antibiotic. Unlike penicillin, tetracycline inhibits bacterial growth intracellularly by binding to the bacterial 30S ribosomal subunit, thus disrupting protein synthesis [20]. The mechanism by which M. oleifera derived isothiocyanates result in bacterial inhibition is not understood to date. However, extensive research documenting the efficacy of synthetic, highly active antibacterial peptides derived from M. oleifera seed proteins has helped elucidate the mechanism by which the cationic peptide results in microbial inhibition [13]. The secondary structure of the antimicrobial polypeptide contains positively charged α- helices that are thought to bind to negatively charged phospholipid heads of the bacterial cell membrane; the charge attraction and stabilization allows for subsequent interaction between a loop region in the antibacterial peptide structure and aliphatic fatty acids of the bacterial membrane [13]. In turn, the bacterial membrane is destabilized by changes in permeability and lipid distribution, as well as disruption of its membrane potential. In sum, these effects result in breaks in the bacterial cell membrane, disaggregation of its components, and eventually cell death [12] [13]. With this understanding, antibiotic peptides of M. oleifera seed extracts were expected to exhibit inhibition comparable to that of penicillin; however, they were not expected to produce results similar to tetracycline. Given that tetracycline works intracellularly as a bacteriostatic antibiotic, it is possible that other constituents of M. oleifera seed 69 www.earthjournals.org Volume 3 Issue 2 2013
  • 10. INTERNATIONAL JOURNAL OF PHYTOTHEARPY RESEARCH ISSN 2278 – 5701 extracts, such as the identified isothiocyanates, also work intracellularly in a concerted effort with the antibiotic peptides. V. Future Research In futures trials it would be desirable to repeat the leaf and seed DI H2O extract experimentation, withholding the 80°C incubation that evaporated the solvent. It is suspected that motility afforded to microbial inhibiting molecules by the presence of DI H2O will allow the molecules to disperse across the agar lawn with greater confluence. Repeated experiments could also rule out the possibility of regrowth by collecting data samples over durations shorter than 24-hrs, i.e. 3-hr, 6-hr, 9-hr, or 12-hr time intervals. Although several of the organisms tested in this study are known to be common colonizers of human skin and gastrointestinal tracts, it is recommended that antibacterial susceptibility tests, using M. oleifera extracts, be performed against pathogenic bacteria endemic to resource-poor settings. Further, it is also suggested that MIC studies be conducted using the crude extraction technique. If the extracts prove to be efficacious inhibitors of pathogenic bacteria, then shelf-life studies should be performed to further qualify this pragmatic extraction technique that it may be used in destitute regions worldwide. CONCLUSIONS This study demonstrated that when M. oleifera seeds and leaves are prepared using very crude methods they can provide, to varying abilities, antimicrobial capabilities comparable to some contemporary remedies against common pathogens that cause human morbidity. This research is a reminder that heroic lengths and modern science are not always necessary to combat antimicrobial pathogens in remote regions where modern medicine is not available. ACKNOWLEDGEMENTS Many thanks to Educational Concerns for Hunger Organization (ECHO), located in Ft. Myers, Florida, U.S.A. and Moringa Farms, located in Sherman Oaks, California, U.S.A. for their abundant guidance and provision of plant part specimens for this and future research endeavors. REFERENCES 1. Caceres, A., O. Cabrera, O. Morales, P. Mollinedo & P. Mendia. 1991. Pharmacological properties of moringa oleifera. 1: Preliminary screening for antimicrobial activity. Journal of Ethnopharmacology. 33: 213-216. 2. Silverstein, K. 1999. Millions for Viagra, Pennies for Diseases of the Poor. The Nation. http:// www.t henation. com/article/millions-viagra-pennies-diseases -poor. (accessed April 2013). 3. Osrin, D., S. Vergnano & A. Costello. 2004. Serious bacterial infections in newborn infants in developing countries. Current Opinion in Infectious Disease. 17:217-224. 4. Anwar, F., S. Latif, M. Ashraf & A.H. Gilani. 2007. Moringa oleifera: A food plant with multiple medicinal uses. Phytotherapy Research. 21:17-25. 5. Fahey, J. W., K. L. Wade, K. K. Stephenson & F. E. Chou. 2003. Separation and purification of glucosinolates from crude plant homogenates by high-speed counter-current chromatography. Journal of Chromatography. 996: 85-93. 6. Foidl, N., H.P.S. Makkar, & K. Becker. 2001. The potential of Moringa oleifera for agricultural and industrial uses. Moringa News. 70 www.earthjournals.org Volume 3 Issue 2 2013
  • 11. INTERNATIONAL JOURNAL OF PHYTOTHEARPY RESEARCH ISSN 2278 – 5701 www.morniganews.org/actes/foidl_en.doc. (accessed April 2013) 7. Price, M. 2007. ECHO Technical Note: The Moringa Tree. The Peace Corps. http://togo.peacecorps.gov/media/the_morin ga_tree.pdf. (accessed April 2013). 8. Fahey, J. W. 2005. Moringa oleifera: A Review of the Medical Evidence for Its Nutritional Therapeutic and Prophylactic Properties. Part 1. Trees for Life Journal. 1:5. 9. Fuglie L. 2001. The Miracle Tree: The Multiple Attributes of Moringa. CTA Publication, Wageningen, The Netherlands. Pp 117-136 in Combating Malnutrition with Moringa. 10. Gopalan C., B.V. Rama Sastri & S.C. Balasubramanian. 1981. Nutritive values of Indian foods. National Institute of Nutrition, Indian Council of Medical Research, Hyderabad, India. 11. Makkar H.P.S. & K. Becker. 1996. Nutritional value and antinutritional components of whole and ethanol extracted Moringa oleifera leaves. Animal Feed Science and Technology. 63:211–228. 12. Suarez, M., J. M. Entenza, C. Doerries, E. Meyer, L. Bourquin, J. Sutherland, I. Marison, P. Moreillon & N. Mermod. 2003. Expression of a plant-derived peptide harboring water-cleaning and antimicrobial activities. Biotechnology and Bioengineering 81:13-20. 13. Suarez, M., N. Mermod & F. Fisch. 2004. Flo antibacterial peptide from the tropical tree Moringa oleifera: A template for novel antibacterial agents. Universite de Lausanne. www.moringanews.org/documents/ flo.pdf. (accessed April 2013). 14. Guevara, A. P., C. Vargas, H. Sakurai, Y. Fujiwara, K. Hashimoto, T. Maoka, M. Kozuka, Y. Ito, H. Tokuda & H. Nishino. 1999. An antitumor promoter from moringa oleifera lam. Mutation Research 440:181- 188. 15. Lipipun V., M. Kurokawa, R. Suttisri, P. Taweechotipatr, P. Pramyothin, M. Hattori & K. Shiraki. 2003. Efficacy of Thai medicinal plant extracts against herpes simplex virus type 1 infection in vitro and in vivo. Antiviral Research. 60:175–180. 16. Tahiliani P. & A. Kar. 2000. Role of Moringa oleifera leaf extract in the regulation of thyroid hormone status in adult male and female rats. Pharmacology Research. 41:319-323. 17. Haristory, X., J. Fahey, I. Scholtus & A. Lozniewski. 2005. Evaulation of the Antimicrobial Effects of Several Isothiocyanates on Helicobacter pylori. Planta Medica. 71:326-330. 18. Price, M. 2006. Using Science to Help the Poor: Low-Budget Research Ideas. Part 3: Research Opportunities. Trees for Life Journal. 1:9. 19. Fuglie, L. 2005. The Moringa Tree: A local solution to malnutrition. Church World Service, Dakar. www.moringanews.org/documents/Nutrition .pdf. (accessed April 2013). 20. Willey, J., L. Sherwood & C. Woolverton. 2009. Prescott’s Principles of Microbiology. McGraw Hill Higher Education, New York. 71 www.earthjournals.org Volume 3 Issue 2 2013