CHAPTER TWO

LITERATURE REVIEW

2.0 INTRODUCTION
Antibiotics have been considered one of the wonder discoveries of the 20th century. This is true
but the real wonder is the rise of antibiotics resistance in hospitals. The extra-ordinary genetic
capacities of microbes have been benefited from man’s overuse of antibiotics to exploit every
source of resistant genes and every means of horizontal gene transmission to develop multiple
mechanisms of resistance for each and every antibiotic introduced into practice clinically. This
review presents the silent aspect of antibiotic resistance over the past century. To achieve
complete restitution of therapeutic application of antibiotics, there is a need for more information
on the role of environmental microbes in the rise of antibiotic resistance. In particular, creative
approaches to the discovery of new antibiotics and their expedited and controlled introduction to
therapy are obligatory (Julian and Dorothy, 2010).

The first ever antibiotic drug to be discovered was penicillin by Alexander Fleming in the 1940s
in London. The discovery of penicillin in 1940s was recognized as one of the greatest advances
in therapeutic medicine. It was discovered in the United Kingdom but due to the World War II,
United States played the major role in developing large scale production of the drug but the drug
subsequently went out of supply (Pfizer and Merck, 1999). Since then, many researchers have
been seeking new sources of antibiotics including plants’ products.
Natural products derived from plants for the treatment of diseases have proven that nature stands
a golden mark to show the relationship between man and his environment (Oladeji et al., 2016).
Plants are important source of medicine and play a key role in world health. The use of medicinal
plants has attained a commanding role in health system all over the world. This involves the use
of medicinal plants not only for the treatment of diseases but also as a potential material for
maintaining good health and conditions. This is due to their better cultural acceptability, better
compatibility and adaptability with the human body and pose little site effects (Oladeji, et al.,
2016).
2.1 BOTANIC DESCRIPTION OF MORINGA OLEIFERA
Moringa Oleifera is a small, graceful, deciduous tree with sparse foliage, often resembling a
leguminous species at a distance, especially when in flower, but immediately recognized when in
fruit. It has a high germination rate of about 85%. The tree grows to 8m high and 60cm. Bole
crooked, often forked from near the base. Twigs and shoots shortly but densely hairy. Crown
wide, open, typically umbrella shaped and usually a single stem; often deep rooted. The wood is
soft.

Leaves alternate, the old ones soon falling off; each leaf large (up to about 90cm long), with
opposite pinnae, spaced about 5 cm apart up the central stalk, usually with a 2nd lot of pinnae,
also opposite, bearing leaflets in opposite pairs, with a slightly larger terminal leaflet. Leaflets
dark green above and pale on the under surface; variable in size and shape, but often rounded-
elliptic, seldom as much as 2.5 cm long.

Flowers throughout the year, in loose axillary panicles up to 15 cm long; individual flower stalks
up to 12 mm long and very slender; 5 pale green sepals 12 mm long, finely hairy, 5 white petals,
unequal, a little longer than the sepals; 5 stamens with anthers, 5 without; style slender. Flowers
very sweet smelling.

Fruits are large and distinctive, up to 90 cm long and 12 mm broad, slightly constricted at
intervals, gradually tapering to a point, 3- (4- ) angled, with 2 grooves on each face, light brown.
It splits along each angle to expose the rows of rounded blackish oily seeds, each with 3 papery
wings.

Readily colonizes stream banks and savannah areas where the soils are well drained and the
water table remains fairly high all the year round. It is quite drought tolerant but yields much less
foliage where it is continuously under water stress. It is not harmed by frost, but can be killed
back to ground level by a freeze. Moringa oleifera Lamarck (fam. Moringaceae), is a perennial
foliaged tree, widely cultivated due to its high adaptability to climatic conditions and dry soils
(Okuda, Baes, Nishjima & Okada, 2011).
It quickly sends out new growth from the trunk when cut, or from the grown when frozen. It is
adapted to a wide range of soil types but does well in well drained clay or clay loam without
prolonged waterlogging. Prefers a neutral to slightly acidic soil reaction, but it has recently been
introduced with success in Pacific atolls where the pH is as high as 8.5. This tree is in the
Moringaceae, or horseradish tree family which is closely related to the papaya containing
Caricaceae family. It is the sole genus, containing thirteen species one such species being M.
stenopetala, a species native to and cultivated in Africa.

Taxonomic Classification

Kingdom - Plantae
Sub-Kingdom -Tracheobionta (vascular plants)
Super-division - -Spermatophyta(seed plants)
Division -Magnoliophyta(flowering plants)
Class - Magnoliopsida(Dicotyledons)
Sub-class -Dillenidae
Order -Cappparales
Family -Moringaceae
Genus -Moringa
Species -Moringa Oleifera

2.1.1 HISTORY OF THE PLANT

The generic name of Moringa oleifera is derived from the Tamil (language spoken in southern
India and northeast Sri Lanka) word ‘Murungai’ meaning twisted pod and ‘Oleifera’ is Latin
meaning ‘oil-bearing’ due to the seeds high oil content.

It is believed that the Moringa tree originated in northern India and was being utilized in Indian
medicine around 5000 years ago, and there are also accounts of it being utilized by the ancient
Greeks, Romans, and Egyptians. This tree was and still is considered a panacea and is referred to
as the “Wonder Tree”, “Divine Tree”, and “Miracle Tree” amongst many others.
It is considered as one of the most useful plant in the world because almost all its parts can be
used as food, in traditional medicines and for industrial purposes (Fahey, 2005; Khalafalla &
Abdellatef, 2010). In addition, seed and leaf flour have been used in the formulation of infant
food to increase protein content (Anwar, Latif, Ashraf & Gilani, 2007).

Moringa Oleifera has been used extensively in traditional medicine for the treatment of several
ailments, promotes digestion, skin diseases, diarrhea, as stimulant in paralytic afflictions,
epilepsy and hysteria (Farooq et al., 2012; Mishra et al, 2011). Various parts of the plant have
been experimentally shown to have anti-inflammatory action (Jamil et al., 2007), the leaves,
stem bark and seeds have been reported to have therapeutic properties (Anwar and Rashid,
2007).

2.1.2 ACTIVE COMPONENTS OF Moringa oleifera AND THEIR PROPERTIES.

Moringa oleifera is highly nutritious plant because all parts of it is a good source of protein,
vitamins, essential amino acids, minerals and various phenolics compounds and moringa leaves
contain negligible content of antinutritional factors such as tannins, saponins, tripsin inhibitors
and phytates (Eman, 2014). Different morphological parts of M. oleifera such as leaves, stems,
whole plants and pods contain various levels of crude protein (71.2-267.9 g kg -1 DM), crude
fiber (210.0-490.0 g kg -1 DM), NDF (48-842 g kg -1 DM), ADF (39-805 g kg -1 DM) and ADL
(11-452 g kg -1 DM) according to plant parts (Mabruk et al. 2010).

Minerals: Mineral compositions of plants differ depending on various factor such as climatic and
soil factors (Offor et al. 2014). It is reported that Moringa oleifera leaves generally contain a
higher amount of minerals compared to seeds (Limmatvapirat et al. 2015). Mineral compositions
of Moringa oleifera leaves and seeds are shown in Table

Minerals Leaves (mg/kg) Seeds (mg/kg)

Calcium 4900 2925

Phosphorus 3600 1600

Potassium 13800 9615

Sodium 6700 7140

Magnesium 2700 2998

Manganese 122 125

Iron 415 142

Zinc 47 41

Copper 12 6

* Limmatvapirat et al. 2015.

As seen on the table above, Moringa oleifera leaves are very rich in minerals. While fresh
Moringa leaves contain 4 times the calcium of milk, 3 times potassium of banana and 3/4 iron of
spinach, dried leaves contain 17 times the calcium of milk, 15 times potassium of bananas and 25
times iron of spinach. Similarly, vitamin contents of Moringa oleifera leaves and seeds are very
rich. Fresh leaves contain 7 times the vitamin C of oranges and 4 times vitamin A of carrots.
However, dried leaves contain 10 times the vitamin A and 1/2 times the vitamin C of oranges
(Mahmood et al. 2010).

2.1.3 MECHANISM OF ACTION OF Moringa oleifera

Analgesic, anti-inflammatory, and antipyretic activities

Almost every part of this “miracle tree” has been found to exhibit analgesic activity in different
animal models. Extract of leaves, seeds, and bark showed significant analgesic activity in both
central (hot plate method) and peripheral models (acetic acid–induced writhing method) in a
dose-dependent manner and extracts of leaves exhibited analgesic potency similar to that of
indomethacin and antimigraine properties in a dose-dependent manner. Topical application
showed efficacy against multiple sclerosis–induced neuropathic pain.
Anti-inflammatory activity of leaf extract has been observed in a carrageenan-induced paw
edema model. Extracts of bark showed anti-inflammatory activity comparable to diclofenac in
the same model. Anti-inflammatory properties of root have also been reported. Mechanism
underlying the anti-inflammatory activity may be attributed to the regulation of neutrophils and
c-Jun N-terminal kinase pathway. Active ingredients contributing to anti-inflammatory property
are tannins, phenols, alkaloids, flavonoids, carotenoids, β-sitosterol, vanillin, hydroxymellein,
moringine, moringinine, β-sitostenone, and 9-octadecenoic acid.

Leaf extract showed significant antipyretic activity in a Brewer’s yeast–induced pyrexia model.
Ethanol and ethyl acetate extracts of seeds also showed significant antipyretic activity.

Neuropharmacological activity

Aqueous extract of leaves has shown protection against Alzheimer’s disease in a colchicine-
induced Alzheimer’s model using behavioral testing (radial Y arm maze task). It protected
against Alzheimer’s disease by altering brain monoamine levels and electrical activity. Another
study using toluene-ethyl acetate fraction of methanolic extract of leaf showed potent nootropic
activity. Leaf extract contains vitamins C and E, which play a significant role in improving
memory in patients with Alzheimer’s disease.

Anticonvulsant activity of leaves was shown in both pentylenetetrazole and maximum electric
shock models using male albino mice. Aqueous extract of root suppressed penicillin-induced
epileptic seizures in adult albino rats.

Ethanolic extract of leaves exhibited both central nervous system depressant and muscle relaxant
activities in actophotometer and rotarod apparatuses, respectively and also exhibited significant
anxiolytic activity in staircase test and elevated plus maze test in a dose-dependent manner.
Anticancer activity

Alcoholic and hydro methanolic extracts of leaves and fruits showed a significant growth delay
in tumor kinetics in mouse melanoma tumor model studies. Extract of leaf also exhibited
antiproliferative activity on A549 lung cells. Exploration of effects on prerequisites for cancer
metastasis showed that the administration of leaf extract into chick chorioallantoic membrane led
to an antiangiogenic effect, which was dose dependent, thereby showing their remarkable
anticancer potential. Another study reported that pod extract suppressed azoxymethane and
dextran sodium sulfate–induced colon destruction in male, Institute of Cancer Research (ICR)
mice. An extract of root and leaf showed a cytotoxic effect against breast cancer,
hepatocarcinoma, and colorectal cancer cells in vitro and cisplatin-resistant ovarian cancer cells.
Flower extracts stimulated cell proliferation in normal cells but not in cancer cells, whereas leaf
extract showed marked antitumor and hepatoprotective effects, these findings suggest the
regenerative potential of MO besides its anticancer effects. Phytoconstituents such as niazimicin,
carbamates, thiocarbamate, nitrile glycosides and others such as quercetin and kaempferol are
responsible for the anticancer activity of this plant.

Antioxidant activity

MO fruits and leaves have antioxidant properties. Extract of leaf showed a concentration-
dependent increase in glutathione level and a decrease in malondialdehyde level, fruit extract
showed beneficial results in eliminating free radicals, extract of roots significantly reduced iron
and FeSO4-induced microsomal lipid peroxidation in a dose-dependent manner. Pods were
capable of scavenging peroxyl, superoxyl, and 2, 2-diphenyl-2-picryl hydrazyl (DPPH) radicals.
Besides displaying antioxidant activity, MO leaf extract also showed a dose-dependent
nephroprotective action in an acetaminophen-induced nephrotoxicity model in male BALB/c
rats. Triterpenoids, moringyne, monopalmitic and di-oleic triglyceride, campesterol,
stigmasterol, β-sitosterol, avenasterol, vitamin A, and its precursor beta-carotene have been
shown to contribute for antioxidant properties.
Effects on the reproductive system

Plant extract showed a significant increase in the weight of testis, seminal vesicle, epididymis,
and a higher score for epididymal maturity and lumen formation along with an increase in
seminiferous tubule diameter (all doses).

Ethanolic extract of plant protected prepubertal spermatogonial cells in Swiss male albino mice
in cyclophosphamide-induced damage model; the possible underlying mechanism may be
upregulation of expression of c-Kit and Oct4 transcripts independent of p53-mediated pathway.

The abortive effect of leaf extract on rats after treatment for 10 days after insemination has been
reported. Extract showed a synergistic effect with estradiol and an inhibitory effect with
progesterone. Fresh leaves of MO contain approximately 11,300–23,000 IU of vitamin A, which
has a major role in various anatomical processes, such as reproduction, embryonic growth and
development, immunity development, and cell differentiation.

Hepatoprotective activity

Extract of plant has shown hepatoprotective effects against carbon tetrachloride and
acetaminophen-induced liver toxicity in Sprague Dawley rats and also hepatoprotective effect
against antitubercular drugs and alloxan-induced liver damage in diabetic rats. This plant-based
diet for 21 days showed significant potential in attenuating hepatic injury. Alkaloids, quercetin,
kaempferol, flavonoids, ascorbic acid, and benzylglucosinolate were found to be responsible for
hepatoprotective activity.

Gastroprotective and anti-ulcer activities

Extract of plant remarkably reduced ulcer index in ibuprofen-induced gastric ulcer model and in
pyloric ligation test and a significant reduction in cysteamine-induced duodenal ulcers and stress
ulcers was also observed. Bisphenols and flavonoids could be contributing to this property.
Cardiovascular activity

Extract of MO plant significantly reduced cholesterol levels and displayed a protective role on
hyperlipidemia induced by iron deficiency in male Wistar rats. Antihypertensive effect of leaf
extract on spontaneous hypertensive rats was shown, in addition to reduced chronotropic and
inotropic effects in isolated frog hearts. Active constituents for hypotensive action are niazinin
A, niazinin B, and niazimicin. Extract of leaves also showed cardioprotective effects against
isoproterenol-induced myocardial infarction in male Wistar albino rats; the mechanism
underlying this cardioprotective activity was found to be antioxidant effect, prevention of lipid
peroxidation, and protection of histopathological and ultrastructural disturbances caused by
isoproterenol. A study was done of Moringa oleifera Lam. on various tissue systems and it
showed reduction in inflammation and lipid accumulation.

Anti-obesity activity

Significant reduction in body mass index was observed after oral treatment with leaf powder
compared with that in obese control. Treatment of hypercholesterolemia rats with methanolic
extract of MO leaf for 49 days showed a remarkable reduction in total cholesterol, triglycerides,
and body weight, moreover, liver biomarkers, organ weight, and blood glucose levels were also
decreased. Mechanisms include downregulation of mRNA expression of leptin and resistin and
upregulation of adiponectin gene expression in obese rats.

Antiasthmatic activity

Extract of seeds showed protection against asthma as investigated in various models; the
proposed mechanism for this effect was a direct bronchodilator effect combined with anti-
inflammatory and antimicrobial actions and inhibition of immediate hypersensitive reaction.
Ethanol extract of seeds tested against ovalbumin-induced airway inflammation in guinea pigs
showed a significant increase in respiratory parameters and reduction in interleukins in
bronchoalveolar lavage.
Hematological activity

A randomized, double-blind, placebo-controlled study was carried out on women who were
anemic with hemoglobin levels between 8 and 12g/dL and were treated with aqueous extract of
moringa plant, the results showed an increase in mean hemoglobin and mean corpuscular
hemoglobin concentration. Another study revealed that when moringa was given to healthy
human volunteers for 14 days, a significant improvement in platelet count was observed.

Antidiabetic activity

Extract of leaf showed significant antihyperglycemic and hypoglycemic activity in normal and
alloxan-induced diabetic rats. An elaborate study was performed to determine the effect of
aqueous leaf extract on lipid profile, body weight, glucose; plasma insulin, homeostatic model
assessment, and oral glucose tolerance test in insulin-resistant (IR) and type 1 diabetic rat
models. IR rats were fed a high-fructose diet, and type 1 diabetic rats were treated with
Streptozotocin (STZ) (55mg/kg). IR rats showed an increase in hyperinsulinemia,
hyperglycemia, and body weight, whereas STZ-induced diabetic rats showed hyperinsulinemia
and hyperglycemia. Leaf extract administration for 60 days returned all the abnormal parameters
to normal levels.

Furthermore, extract of leaf inhibited the formation of advanced glycation end products by
reducing monosaccharide-induced protein glycation. Glucomoringin, phenols, flavonoids,
quercetin-3-glucoside, fiber, and phenol have been reported to be responsible for antidiabetic
activity

Anti-urolithiatic activity

Aqueous and alcoholic extracts of this plant showed anti-urolithiatic activity in a hyperoxaluria-
induced rat model and in ethylene glycol–induced urolithiasis model.

Diuretic activity

Leaves, flowers, seeds, roots, and bark extracts increased urine output in rats, extract of leaf
showed a dose-dependent diuretic action greater than control but less than hydrochlorothiazide.
Campesterol, stigmasterol, β-sitosterol, and avenasterol were responsible for this activity.
Anti-allergic activity

Ethanolic extract of seeds inhibited passive cutaneous anaphylaxis induced by anti-

immunoglobulin G (IgG) antibody and histamine release from mast cells; the mechanism
underlying this action could be membrane-stabilizing action and also reduced scratching
frequency in an Ovalbumin sensitization model.

Anthelmintic activity

This plant showed potent anthelmintic activity, it took less time to paralyze Indian earthworm
Pheretima posthuma. In ovicidal assay, ethanolic and aqueous extracts showed 95.89% and
81.72% egg hatch inhibition, respectively, and in larvicidal assay, they showed 56.94% and
92.50% efficacy, respectively.

Wound-healing activity

Extracts of leaf, dried pulp, and seeds showed a significant increase in hydroxyproline content,
wound-closure rate, granuloma-breaking strength, and granuloma dry weight, and a decrease in
scar area and skin-breaking strength in incision, excision, and dead space wound models in rats.

Studies conducted on the effect of wound healing of leaf extract in diabetic animals showed
improved tissue regeneration, decreased wound size, downregulated inflammatory mediators,
and upregulated vascular endothelial growth factor in wound tissues and remarkable
antiproliferative and anti-migratory effects on normal human dermal fibroblasts.

Antimicrobial activity

Ethanolic extract of leaf showed antimicrobial activity against all the tested bacteria. Chloroform
extract reported activity against pathogens such as Salmonella typhi, Pseudomonas aeruginosa,
Escherichia More Details coli, and Vibrio cholera.
Ethanolic extracts of root and bark possessed antifungal activity against Aspergillus niger,
Neurospora crassa, Rhizopus stolonifer, and Microsporum gypseum and also showed inhibitory
activity against Leishmania donovani. Many studies suggest that extracts of seeds could be a
potential option to purify water sources as it inhibited bacterial growth in agar and nutrient
medium. Methanolic extract of leaves inhibited urinary tract pathogens, such as Staphylococcus
aureus, Klebsiella pneumoniae, S. saprophyticus, and E. coli.

Flavonoids, tannins, steroids, alkaloids, saponins, benzyl isothiocyanate, and benzylglucosinolate

were found to be responsible for antimicrobial activity, whereas pterygospermin was found to be
responsible for antifungal activity.

Immunomodulatory activity

Methanolic extract of this plant stimulated both humoral and cellular immune response. In
addition, extract showed an increase in optical density and stimulation index, indicating
splenocyte proliferation.

Antidiarrheal activity

Extract of seeds showed significant reduction in gastrointestinal motility and were found to be
effective in castor oil induced diarrhoea in male Wister rats. Antidiarrheal activity can be
attributed to phytochemical ingredients such as tannins, saponins, and flavonoids.

Miscellaneous effects

Extract of this plant exhibited a reduction in unwanted sebum secretions from sebaceous gland
during winter in humans. A systematic review and meta-analysis have clearly accounted this
plant as a galactagogue. Methanolic extract of root showed local anesthetic action in frog and
guinea pig models. Significant CYP3A4 inhibitory effects were exhibited by MO extract. Thus,
MO has a great potential for herb–drug interactions.
2.1.4 CLINICAL USES OF Moringa oleifera

In the recent years, there is a demand for natural foods that contain active substances such as
phytochemicals (Godinez-Oviedo et al. 2016). The different parts of Moringa oleifera are good
source of various phytochemicals such as alkaloids, flavonoids, phenolics, glucosinolates,
carotenoids, sterols (Valdez-Solana et al. 2015). These phytochemicals bring healing properties
to Moringa oleifera (Gopalakrishnan et al. 2016). Flavonoids, tannins, steroids, alkaloids,
saponins, benzyl isothiocyanate, and benzylglucosinolate were found to be responsible for
antimicrobial activity, whereas pterygospermin was found to be responsible for antifungal
activity. It is reported that Moringa oleifera has many positive effects like anti-oxidant (Vongsak
et al. 2013), anti-tumor (Khalafalla et al. 2010), anti-inflammatory (Rajanandhet al. 2012), anti-
diabetic (Jaiswal et al. 2009), anti-bacterial (Moyo et al. 2012b), hypolipidemic (Sangkitikomol
et al. 2014), immunomodulatory (Sudha et al. 2010), hepatoprotective (Al-Said et al. 2012).Some
medicinal uses of different parts of Moringa oleifera include:
Leaves
Moringa leaves treat asthma, hyperglycemia, dyslipidemia, flu, heart burn, syphilis, malaria,
pneumonia, diarrhea, headaches, scurvy, skin diseases, bronchitis, eye and ear infections. Also
reduces, blood pressure and cholesterol and acts as an anticancer, antimicrobial, antioxidant,
antidiabetic and antiatherosclerotic agents, neuroprotectant.
The presence of flavanoids gives leaves the antidiabetic and antioxidant properties. The
isothiocyanates are anticancer agents. Flavanoids like quercitin and others are known for
antiproliferative, anticancer agent. The presence of minerals and vitamins help in boosting the
immune system and cure a myriad of diseases.
Seeds
Seeds of moringa help in treating hyperthyroidism, Chrohn’s disease, anti-herpes-simplex virus
arthritis, rheumatism, gout, cramp, epilepsy and sexually transmitted diseases, can act as
antimicrobial and antiinflammatory agents.
The presence of flavanoids gives its antiinflammatory property. The antibiotic pterygospermin is
responsible for antimicrobial properties. The other phyto-chemicals help in treating various
diseases.
Root Bark
Root bark acts as a cardiac stimulant, antiulcer and antiinflammatory agent.
The alkaloid helps the bark to be antiulcer, a cardiac stimulant and helps to relax the muscles.
Flower
Moringa flowers act as hypocholesterolemic, anti- arthritic agents can cure urinary problems and
cold.
The presence of nectar makes them viable for use by beekeepers.
Pods
Moringa pods treat diarrhea, liver and spleen problems, and joint pain.
The presence of PUFA in the pods can be used in the diet of obese.
**Adopted from Gopalakrishnan et al. 2016.

2.1.5 SITE EFFECTS OF Moringa oleifera

Moringa has laxative properties. In large quantities, it can cause stomach upsets, gaseous
distension, diarrhoea and heartburn. If you don't like the taste, it may activate your gag reflex.
Moringa is traditionally used to abort pregnancies in the early stages. Pregnant women should
therefore avoid moringa.

This is a list of interactions that have been observed so far, but it is by no means meant to be an
exhaustive list. To avoid adverse effects or unexpected interactions, talk to your doctor before
starting a moringa supplement.

- Since moringa does affect the CYP450 enzymes through which most drugs are
metabolized, other drug interactions are possible. Drugs that could be especially affected
include statins, anti-seizure and antifungal medications
- Moringa also reduced blood sugar levels, which may dangerously reduce blood glucose
levels in people with type II diabetes.
2.2. CHEMICAL CONSTITUENTS OF PLANTS AND THEIR IMPORTANCE.
The use of plants for treating diseases is as old as the human species. Flavonoids have been
found to exhibit greater antifungal and antibacterial activities against some human pathogenic
bacteria and fungi. Medicinal plants contain a variety of secondary metabolites such as tannins,
terpernoids, alkaloids, flavonoids that dictate the therapeutic potency of the plants most
especially the antimicrobial properties (Oladeji O 2016).
The tannin present in medicinal plants makes it useful in the production of antiseptic soaps
which are commonly used in bathing. It has also been documented that phytochemicals can be
toxic to filamentous fungi, yeast and bacteria and also inhibitory to viral reverse transcriptase.
Saponins has been reported as a major component acting as antifungal secondary metabolites. A
wide range of physiological activities of saponins, phenols, steroids and tannins are found to be
more predominant and they may be responsible for antimicrobial action (Oladeji O et al., 2016).
Despite the numerous beneficial effects of plants constituents that have been known worldwide,
there are also adverse effects of these plants constituents.
Irritants contact dermatitis caused mechanically by spines, irritants hairs or irritant chemicals in
sap.
Phytophotodermatitis resulting from skin contamination by plant containing furoumarins and
subsequent exposure to UV light.
Immediate (type 1) or delay hypersensitivity contact reactions mediated by the immune system
in individual sensitized to plants or plants products.

2.3. SOME PLANTS OF MEDICINAL IMPORTANCE

A number of plants have been used in traditional medicine for many years now. According to
WHO, 2010, medicinal plants will be the best source to obtain a variety of drugs for treating
infectious diseases. Biochemical compounds are synthesized and stored in these plants in the
form of phenolics and phytochemicals. Moreover, Many plants (like those listed below) exhibit
antimicrobial activity against common human pathogens such as Klebsiella pneumonia,
Salmonella typhi, B.ceurus and E. coli (Anjana et al., 2009).
Some plants contain fungi such as Trichilia elegans which when isolated proved to exhibit
antibacterial activity against human pathogens such as P. aerogenosa (Rhoden et al., 2012).
Others like Aloe vera have been traditionally used in the treatment of infectious diseases such as
cough, ulcers without a scientific notion whether it truly possess antibacterial properties
(Priyanka et al., 2013).
Below is a list of some plants of medicinal importance and the disease conditions they can
intervene for.
Table 2: List of Some Plants of Medicinal Importance

Scientific Name Part of plant Disease conditions (… etc.)

Moringa oleifera Whole plant Arthritis, cancer, diarrhoea, abscesses
Aloe vulgaris Whole plant Rashes; ulcers, burns
Allium sativum Cloves, roots Reduce LDL and Cholesterol levels
Carica papaya Leaves, seeds Jaundice, pinworm and roundworm
Bryophillum pinnatum Leaves Wound healing, influenza
Cassia alata Leaves Dermatosis, wound infections
Centella asiatica Leaves Cellulitis, chronic liver disease
Citrus limon Fruit Arteriosclerosis, gastritis,
Zingiber officinale Rhizome Sore throat, coughing
(Jamshidi et al., 2018)
2.4. PHYTOCHEMICALS PRESENT IN VEGETATIVE PLANTS

2.4.1 Flavonoids

Flavonoids are important group of polyphenols, made up more than one benzene ring in their
structure. They protect plants from biotic and abiotic stresses agents and act as UV filter,
function as detoxifying agents and antimicrobial defensive compounds. They absorb the harmful
UV radiation, induce cellular damage and also influence the transportation of plant hormone;
auxins (Samanta et al., 2011).

Flavonoids have been reported to exert a wide range of biological activities which include;
antioxidant effect, antimicrobial effect, anti-inflammation, cardio-protective effects, anti-
carcinogenic effects and many others. Their mode of action is related to their ability to inactivate
microbial adhesions, cell envelop transport proteins and others (Kalita et al., 2011).

Figure I: Chemical Composition of Flavonoids

2.4.2 Saponins

Saponins are glycosides of Terpenes and steroids. They are sometimes known as steroidal
glycoalkaloids. The structural diversity of Saponins is reflected in the array of different
biological activities, associated with these compounds and these diverse compounds provide a
significant resource for drugs and agro-chemical discovery (Reddy et al., 2013). Saponins
collectively have a wide range of biological, antifungal, anti-inflammatory, and antimicrobial
activity and so many have roles in plant defence (Podolak et al., 2010)
They are typically composed of hydrophobic substance which is extensively decorated with
functional group prior to the addition of hydrophilic sugars. Different type of saponins have been
isolated from roots, stems, leaves, fruits and flowers of the plant. The leaves are the first organs
to attain maximum concentration of saponins, followed by unripe fruits then flowers. Saponins
have many health benefits which include reduction of cholesterol level, immunity booster,
reduce bone loss and act as an antioxidant (Reddy et al., 2013).

Figure II: chemical structure of Saponins

2.4.3 Tannins

Tannins are water-soluble phenolic compounds with molecular weight ranging from 500 – 3000
that have the property of combining with proteins and cellulose to form insoluble complex. They
are derivatives of flavones. They contain tannic acid and its chemical structure depends on the
plant species producing it. All tannins have some common features which enable their
classification into two main groups; three types of hydrolysable tannins (gallotannins,
ellagitannins and complex tannins) and the condensed tannins (non-hydrolysable) called
procyanidins. Tannins act as chelating agents by reducing the availability of essential metal ions
for microorganisms, biological antioxidant depending on its concentration. The mode of
antimicrobial action of tannins appears to be related to the inhibition of cellular microbial
enzymes (cellulose, pectinase and glycosyl transferase). The antimicrobial activity of tannins is
also related to their action on the microbial membranes (Preeti et al., 2016).
TANNIN
S

Hydrolysable Tannins Condensed Tannins

Figure III: Chemical composition of Tannins

2.4.4 Alkaloids

Alkaloids are a group of naturally occurring chemical compound that mostly contain basic
nitrogen atom in a heterocyclic ring. Like plant hormones, they function as plant stimulant or
regulators in activities such as growth, metabolism and reproduction.
Alkaloids are colourless, crystalline, non-volatile solids; few such as Coniine and Nicotine are
liquid and a few are colourless. The free bases alkaloids are soluble in water and diluted alcohol
but insoluble in most organic solvents. Although many of the alkaloids possess curative
properties and are of great value in medicine, they are powerful poison (Arya et al., 2012).

Figure IV: chemical structure of alkaloid

2.4.5 Some Bacteria and Fungi Species Sensitive to Phytochemicals

Many bacteria and fungi are sensitive to phytochemicals extracted using appropriate extraction
methods and solvents. Some of these bacteria and fungi include Staphylococcus epidermidis,
Enterococcus faecali, Salmonella paratyphi, Staphylococcus aureus, Escherichia coli,
Mycobacterium tuberculosis, Shigella dysenteriae, Candida albicans (Arulmozli and Microb,
2018).

2.5. MECHANISM OF ACTION OF ANTIMICROBIAL AGENTS

A phytochemical is a biological substance found in plants and make up its active components.
Activities of phytochemicals are enhanced by their properties. Antimicrobials must thus reach
the target sites in sufficient quantities and it must not be inactivated or modified by the
microorganism and a suitable target site must exist in the bacteria cell wall. Phytochemicals act
in the following way;
 a) Inhibition of cell wall synthesis
Inhibition of cell wall synthesis can selectively damage bacteria and fungi. Some phytochemicals
contains β-lactam that prevents cross-link in peptidoglycan which affects mostly active growing
cells; thus inhibiting cell wall synthesis. Resistance arises when there is failure to cross the cell
membrane. Others act by either inhibiting the synthesis of peptidoglycan of disrupting the
movement of peptidoglycan precursors (Onishi et al 2011).
b) Inhibition of protein synthesis
Because enzymes and cellular structures are mostly made of proteins, they are essential for the
survival and multiplication of most bacteria cells. So, phytochemicals (Flavonoids and Tannins)
and antibacterial agents targets bacteria protein synthesis by binding to the 30s and 50s subunits
of the intracellular ribosome. Others act by blocking tRNA from binding to 30s ribosome mRNA
complex. Some even bind to 50s ribosome thereby blocking peptide elongation (Ferrero er et al.,
2009).
c) Inhibition of cell metabolism
These types of antimicrobial agents that inhibit cell metabolism are called antimetabolites. These
compounds inhibit the metabolism of the organism but not that of its host. They do this by
inhibiting enzyme-catalysed reactions which take place in bacterial cell but not in animal cells
(Mandko et al., 2011).
d) Alteration of cell membranes
Some antimicrobial agents act by disrupting cytoplasmic membranes. Resistance arises due to
the inability to penetrate the outer membrane. Some inhibit the synthesis of acids such as folic
acid (Soussy et al., 2010).
e) Inhibition of nucleic acid synthesis
A group of phytochemicals act by interfering with the activity of enzymes such as DNA gyrase
required for supercoiling of DNA. Some instead disrupt DNA structure; inhibit RNA
transcription while others act by binding to DNA dependent RNA polymerase (Pathogenic
Microbiology, 2010).
2.5.1 IMPORTANCE OF MEDICINAL PLANTS
A medicinal plant is any plant which in one or more of its organs, contains substances that can
used for therapeutic purpose or substances that are precursors for synthesis of useful drugs.
Medicinal plants have been used in healthcare since time immemorial, thus play a vital role in
disease prevention, promote healing of injuries and alleviate pains. All over the world, drive to
lower the cost of healthcare has made herbals and botanicals an attractive alternative to more
synthetic remedies (Pennyvama press, 2015).
Awareness and application of plants to prepare food and medicine have been realised through
trial and error and gradually, humans became able to meet their needs from their surroundings.
Medicinal plants are used as medicinal source in almost all cultures. Thus, ensuring the safety,
quality and effectiveness of medicinal plants and herbal drugs has become a key issue in
industrialized and developing countries. Herbal drugs can help the emergence of a new era of the
healthcare system to treat human diseases in the future through the evaluation and
standardization of health of active plant derived compounds. So, knowledge on herbal and
medicinal plants can play a key role in the exploitation and discovery of natural plant resources
(Salim et al., 2009).
From the study carried out by Ajibade et al., 2013 on the phytochemical screening and toxicity
studies on the methanol extract of moringa seeds, it was concluded that the extract of seeds of
M. oleifera is safe both for medicinal and nutritional uses, whereby the methanol extract was
screened phytochemically for its chemical components and used for acute and sub-acute toxicity
studies in rats. The phytochemical screening revealed the presence of saponins, tannins, terpenes,
alkaloids, flavonoids, carbohydrates, and cardiac glycosides but the absence of anthraquinones.
Although signs of acute toxicity were observed at a dose of 4,000 mg kg-1 in the acute toxicity
test, and mortality was recorded at 5,000 mg kg-1, no adverse effect was observed at
concentrations lower than 3,000 mg kg-1. The median lethal dose of the extract in rat was 3,873
mg kg-1. Sub-acute administration of the seed extract caused significant (p<0.05) increase in the
levels of alanine and aspartate transferases (ALT and AST), and significant (p<0.05) decrease in
weight of experimental rats, at 1,600 mg kg-1.

2.6. MEDICINAL PLANT EXTRACTION

Extraction is a process that involves the separation of medicinal action portions (active
ingredient) of plants or animal tissues from the inactive or inert components using selective
solvents. In these procedures, the products obtained from plants are relatively impure liquids,
semi-solids or powders intended for oral or external use (Handa et al., 2012).
These include; classes of preparations known as decoctions, infusions, fluid extracts, semi-solid
extracts and or powdered extracts.
The purposes of standardized extraction procedures for crude drugs are to obtain the
therapeutically desired portions and to eliminate the inert materials by treatment with a selective
solvent. The extract thus obtained may be used as a medicinal agent in the form of powdered or
liquid extracts, or further processed to be incorporated in any dosage form such as tablets or
capsules. It may also be fractionated to isolate individual chemical entities which are used as
modern drugs (Azwanida et al., 2015).

2.6.1 Pre-Extraction Preparation of Plant Sample

The initial stage in studying medicinal plants is the preparation of samples to preserve the
biomolecules in the plant prior to extraction. Plant samples such as leaves, barks, seeds, roots
and flowers can be extracted from fresh or dried plant material. Other pre-preparation of plant
materials such as grinding and drying also influences the preservation of phytochemicals in the
final extract (Sulaiman et al., 2011).
Fresh versus Dried samples
Both fresh and dried sample is used in medicinal plant studies. In most cases, dried sample is
preferred considering the time needed for experimental design. The freshness of the samples
should be maintained to the maximum level as fresh samples are fragile though tend to
deteriorate faster than dried samples. Furthermore, dried samples tend to have higher content of
Flavonoids (Vongsak et al., 2013).

Grinded versus Powered samples

Lowering particle size increases surface contact between samples and the extraction solvents.
Grinding results in coarse smaller samples meanwhile powdered samples have a more
homogenised and smaller particles leading to better surface contact with extraction solvent.
Particle size is a major factor when using enzyme assisted extraction (Borhan et al., 2013).
Air-drying and freeze-drying of plant sample
Air-drying usually takes from 3-7days to months and up to a year depending on the type of
samples dried (leaves or seed). The plant samples are expose to air at ambient temperature. This
drying method does not force dry plant material using high temperature; hence heat labile
compounds are preserved. However, air-drying takes longer time in comparison to microwave
drying and freeze drying. Freeze-drying is a method base on the principle of sublimation where a
solid is changed into a gas phase without entering the liquid phase (Azwanida et al., 2015).

2.7. SOME TRADITIONAL METHODS OF MEDICINAL PLANT EXTRACTION

Macerations
In this method of extraction, coarsely powdered crude drug is placed in a closed container with
the solvent and allowed to stand at room temperature for a period of at least 3 days with frequent
agitation until the soluble matter has dissolved. The mixture is then strained, the marc (the damp
solid material) pressed and the combine liquid clarified by filtration or decanting after standing.
There is another form of maceration; Digestion in which gentle heat is used during the process of
extraction and it is used when moderately elevated temperatures is not objectionable.
In the extraction of Psidium guajava leaves using chloroform, ethanol, methanol and
hydroalcohol, ethanol resulted in highest extraction yield with maximum presence of
phytochemicals such as Saponins, tannins, alkaloids and flavonoids. Non-polar solvent such as
chloroform showed no active compounds preserved (Arya et al., 2012).
Alteration in temperature and choice of solvent enhance the extraction process and a reduction in
the volume needed for the extraction can be introduced in the maceration when such alteration is
not objectionable.
Influential factors such as:
-Solvent type
-Solvent strength and polarity
-Extraction time
-Temperature
-Speed of agitation
-Sample-solvent ratio (1:10w/v) are some of the factors that can influence extraction (Oancea et
al., 2012).
Percolation
This procedure is used most frequently to extract active ingredients in the preparation of tinctures
and fluid extracts. A percolator (cone-shaped vessel open at both ends) is generally used. Solid
ingredients are moistened with an appropriate amount of specified menstruum and allowed to
stand for approximately 4 hours in a well closed container, after which the mass is packed and
the top of the percolator is closed. Additional menstruum is added to form a shallow layer above
the mass and the mixture is allowed to macerate in a closed percolator for 24 hours. When the
percolate measures about three quarters of the required volume, the mixture liquid is clarified by
filtration or by standing followed by decanting (Vongsak et al., 2013).

Hot continuous extraction (Soxhlet extraction).

Here, finely ground sample is placed in a porous bag, made from a strong filter paper or cellulose
which is placed in the chamber of soxhlet apparatus. The extraction solvent is heated in the
bottom of the flask, vaporizes into the sample chamber, condense in the condenser and drip back.
When the liquid content reaches the siphon arm, the content emptied into the bottom flask again
and the process is continued (Vongsak et al., 2013).
This method is considered not environmental friendly and may contribute to pollution problem
compared to advanced techniques such as superficial fluid extraction (SFE) and (MAE).
Microwave assisted extraction (MAE) utilizes microwave energy to facilitate partition of
analytes from the sample matrix into the solvent. Microwave radiation interact with dipoles of
polar and polarizable materials, causes heating near the surface of the materials and heat is
transferred by conduction. In non-polar solvents, poor heating occurs as the energy is transferred
by dielectric absorption only. (Kumoro et al., 2015).

2.8 SOME EXTRACTION SOLVENTS

In order to determine the active compounds found in plants, the choice of solvent used is very
important as a good solvent should have low toxicity, easy to evaporate, have preservative
properties and should promote rapid absorption of the extract. Some factors such as rate of
extraction, quantity to be extracted and diversity of different solvents may affect choice of the
solvent used (Vongsak et al., 2013).
2.8.1 Water
It is a universal solvent, with chemical formula; H2O and it is used to extract non-polar
antimicrobial active products. Plant extracts containing active products such as anthocyanin will
have little effect because it is water soluble. Aqueous extracts contain polyphenol oxidase which
will degrade polyphenol in water extract (Douglas et al., 2013).
In the extraction of Garnicia atriviridis, aqueous extract showed to preserved only traces of
tannins but no trace of alkaloids (Al-Mansoub et al., 2014).

2.8.2 Ethanol
It is a clear, colourless liquid but in more concentrated solutions, it has a burny taste. Its chemical
formula is CH3CH2OH and has a density of 0.789g/mL at 20°C. Its low freezing point has made
it useful as the fluid in thermometers for temperatures below 40°C. Ethanol is made by
fermentation of sugar due to the action of the enzyme; zymase on yeast to produce CO2 and
ethanol (Shahashiri et al., 2009).
High performance of ethanolic extract can be due to the presence of greater amount of
polyphenols extracted by this solvent. Ethanol extracts are more performant in cell wall and seed
degradation with polar character that will favour polyphenol release. By decreasing the
concentration of ethanol, its polarity increases, leading to high amount of the bioactive
flavonoids extracted. Also Ethanol diffuses easily in the cell membrane to extract intra-molecular
components (Quy et al., 2013).

2.8.3 Methanol
It is more polar but highly cytotoxic and this property can sometimes leads to uncertain
outcomes. Methanol is a hydrocarbon composed of carbon, hydrogen and oxygen with the
chemical formula; CH3OH. It is a colourless, neutral polar, highly volatile and flammable liquid.
It is also called methyl alcohol or methyl hydrate (Technical Information and safe Handling
guide for methanol, 2010).
It is miscible in water, ethanol, ether, benzene and ketones. It is capable of extracting both
hydrophilic and hydrophobic molecules from plants. Certain groups of non-polar compounds are
fairly soluble in methanol but most polar phytochemicals are easily isolated with methanol.
(Kumar et al., 2018).
2.9 HUMAN PATHOGENIC BACTERIA AND FUNGI

2.9.1 Escherichia coli

It belongs to the family Enterobacteriaceae and lives in the GIT of humans and animals. It is
therefore considered as an indicator organism for faecal contamination of food and water. It
commonly causes UTIs, but it is the second most common cause of neonatal meningitis after
Streptococcus agalactiae. Intestinal infections are cause by the serotypes of E. coli
(Cheesbrough, 2006).
It is a Gram negative straight rod with peritricous flagella. It is an aerobe that grows at
temperature range of 36-37°C, producing colonies which are 1-4mm in diameter, grey-white and
lactose fermenting though some strains do not ferment lactose at all. It is Indole positive, methyl
red positive, citrate negative, Voges Proskauer negative, motile while some are haemolytic, and
E. coli also produces gas on KIA with no H2S production (Abigail et al., 2013).

2.9.2 Staphylococcus aureus

It belongs to the family Micrococcaceae. It is the only genus of medical importance in this
family. It is a Gram-positive coccus and found as a normal flora on the human body. It is
catalase and oxidase positive. It is the highest bacteria known to cause food poisoning (Monica
Cheesbrough, 2010). S. aureus also causes boils, impetigo, infection of wound, ulcers and burns.
S. aureus is sensitive to flucoxacillin, tetracycline, vancomycin and cephalosporins
(Cheesbrough, 2010).

2.9.3 Candida albicans

Candida albicans is the commonest cause of Candidiasis. The yeast is a common commensal of
the gastro-intestinal tract. Most candida infections are opportunistic occurring in debilitated
persons. Candidiasis is also associated with prolonged broad spectrum antibiotic therapy.
Candida yeast cells can be detected in unstained wet preparation or Gram stain preparation of
skin, urine, vaginal discharge or other exudates from mucosal surfaces. Candida albicans is
sensitive to Nystatin and cotrimazole.
2.10 ANTIMICROBIAL SUSCEPTIBILITY TESTING

2.10.1 Disc Diffusion Techniques

Disc diffusion technique is a method whereby antimicrobial agents of a specific concentration,
diffuses from disc, tablets or strips into the solid culture medium that has been inoculated with
the selected colonies isolated in a pure culture. It is based on the determination of an inhibition
zone which is proportional to the bacterial susceptibility to the antimicrobial agent present in the
disc. When the concentration of the antimicrobial agent becomes so diluted that it can no longer
inhibit the growth of the test organism, the zone of inhibition is demarcated.
The diameter of the zone of inhibition around the antimicrobial disc is related to the MIC for that
particular organism or bacterial isolate. The larger the zone of inhibition, the lower the
concentration of antimicrobial required to inhibit the growth of the organisms, though this may
also depend on the concentration of antimicrobial in the disc (Ashish et al., 2011).

2.10.2 Broth Diffusion Technique

This is a technique in which a suspension of bacteria of an appropriate concentration is tested
against varying concentrations of an antimicrobial agent in a liquid medium of pre-determined
volume. The aim is to determine the lowest concentration of the antimicrobial agent being used
that will inhibit the visible growth of the bacterium under investigation.
The true MIC is the point between the lowest test concentration that inhibit growth of the
bacterium and the next lower test concentration. It can be performed either in tubes containing a
minimum volume of 2mL (macro dilution) or in smaller volumes using micro titration plates;
micro dilution (Preeti et al., 2012).

2.10.3 Agar Dilution Technique

This technique involves the incorporation of varying concentration of an antimicrobial agent into
an agar medium, and then followed by the application of a defined bacterial inoculum to the agar
surface. The results obtained by this method are often considered as the most reliable for the
determination of an MIC for the test bacterium or isolate.
The advantage of this method include; the ability to test multiple bacteria except those that
swarm on the same set of agar plates at the same time and also to an extent, improve the
identification of MIC end point and extend the antimicrobial concentration range.
There is equally the possibility to semi-automate the method using an inoculums-replicating
apparatus as commercially produced inoculum replicators are available now (CMI, 2019).

2.11. MINIMUM INHIBITORY CONCENTRATION (MIC)

The Minimum Inhibitory Concentration (MIC) is defined as the lowest concentration of an
antimicrobial ingredient or agent that is bacteriostatic (prevents the visible growth of bacteria).
MICs are used to evaluate the antimicrobial efficacy of various compounds by measuring the
effect of decreasing concentrations of antibiotic/antiseptic over a defined period in terms of
inhibition of microbial population growth. These evaluations can be quite useful to determine
appropriate concentrations required in the final product, as the concentration of drug required to
produce the effect is normally several hundred to thousands of times less than the concentration
found in the finished dosage form.
Various concentrations of the compounds are inoculated with cultured bacteria, and the results
are measured using agar dilution or broth dilution (macro or micro) to determine at what level
the MIC endpoint is established. Susceptibility testing is typically conducted using organisms
that contribute to an infectious process warranting antimicrobial chemotherapy. A commonly
used cocktail of bacteria is known as the ESKAPE pathogens (Enterococcus faecium,
Staphylococcus aureus, Klebsiella pneumoniae, Acinetobacter baumannii, Pseudomonas
aeruginosa, and Enterobacter species). The ESKAPE pathogens are considered the leading
cause of nosocomial (hospital-related) infections and are known to be resistant to antimicrobial
products. Other organisms such as aerobic or anaerobic bacteria, yeasts, or filamentous fungi,
either alone or in multiple combinations can also be utilized.

2.12. MINIMUM BACTERICIDAL CONCENTRATION

The Minimum Bactericidal Concentration (MBC) is the lowest concentration of an antibacterial
agent required to kill a bacterium over a fixed, somewhat extended period, such as 18 hours or
24 hours, under a specific set of conditions. It can be determined from the broth dilution of MIC
tests by subculturing to agar plates that do not contain the test agent. The MBC is identified by
determining the lowest concentration of antibacterial agent that reduces the viability of the initial
bacterial inoculum by a pre-determined reduction such as ≥99.9%. The MBC is complementary
to the MIC; whereas the MIC test demonstrates the lowest level of antimicrobial agent that
greatly inhibits growth, the MBC demonstrates the lowest level of antimicrobial agent resulting
in microbial death. In other words, if a MIC shows inhibition, plating the bacteria onto agar
might still result in organism proliferation because the antimicrobial did not cause death.
Antibacterial agents are usually regarded as bactericidal if the MBC is no more than four times
the MIC. MBC testing can be a good and relatively inexpensive tool to simultaneously evaluate
multiple antimicrobial agents for potency. The MBC test can be used to evaluate formulation
problems wherein the formulator suspects that the active ingredient is being “bound up” by other
ingredients. The theory is that the MBC will be worse for a formula that has a portion of its
active ingredient chemically combined with other ingredients, thus not available to kill
microorganisms in the suspension.
The Clinical and Laboratory Standards Institute (CLSI) has established protocols and standards
for establishing MIC and MBC in products. A common methodology utilized for MIC is CLSI
M07-A9, Methods for Dilution Antimicrobial Susceptibility Tests for Bacteria That Grow
Aerobically. CLSI also has developed methods specific for the yeasts, filamentous fungi, and
anaerobic bacteria. For MBC determination, CLSI M26-A, Methods for Determining Bactericidal
Activity of Antimicrobial Agents, is an accepted industry standard.