2.1 PLANT PROFILE OF RICINUS COMMUNIS -...
Transcript of 2.1 PLANT PROFILE OF RICINUS COMMUNIS -...
CHAPTER -2 LITERATURE REVIEW
Phytochemical and pharmacological investigation of anti-tubercular activity of the certain indigenous plants
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2.1 PLANT PROFILE OF RICINUS COMMUNIS LINN.
COMMON NAMES: Castor bean, Ricin, Castor oil plant, Palma christi, Wonder tree
2.1.1 TAXONOMICAL CLASSIFICATION:
Ricinus communis Linn.
Kingdom: Plantae -Plants
Subkingdom: Tracheobionta - Vascular plants
Superdivision: Spermatophyta - Seed plants
Division: Magnoliophyta - Flowering plants
Class: Magnoliopsida – Dicotyledons
Subclass: Rosidae
Order: Euphorbiales
Family: Euphorbiaceae - Spurge family
Genus: Ricinus L. – Ricinus
Species: Ricinus communis Linn.(1,2)
.
2.1.2 SYNONYMS:(3)
Table no. 10: Data showing synonyms of Ricinus communis Linn. in different
languages
Sanskrit. Gandharva-Hasta,
Panchi´gul, Vitiri Kannad Harlu
Assam Erri Malylam Ambanakka, Avanakku
Bengali. Bherenda Marathi Erand, Erandee
English Castor Oil Plant Orissa Bheranda
Gujrathi Erando Punjabi Erand
Hindi. Erand, Rendee, Andu Tamil Amanakku
Urdu Erand Telgu Amudanu, Amudmuchetu
2.1.3 DERIVATION OF THE NAME CASTOR:
It is interesting to trace the origin of the name "Castor." Castor is the generic name
of the North American beaver (Castor canadensis) and one of the brightest double stars in
the constellation Gemini. In Greek and Roman legend, castor was one of the twin sons of
Jupiter and Leda. According to E. A. Weiss, writing in castor, Sesame and Sunflower
(1971), the name "castor" has nothing to do with luminous stars, or offspring of Greek and
Roman Gods. Castor was apparently coined by English traders who confused it with the
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oil of another shrub, Vitex agnus-castus, which the Spanish and Portuguese in Jamaica
called "agno-casto." Although it is commonly known as the castor bean plant, the seed is
really not a true bean and it is not related to the bean or legume. There are many other
examples of "beans" that are technically not beans, such as Mexican Jumping "beans" and
coffee "beans".
The scientific name for the castor plant, Ricinus communis, has a much more
logical derivation. Communis means common in Latin, and castor plants were already
commonly naturalized in many parts of the world when the eighteenth century Swedish
naturalist Carolus Linnaeus (Karl von Linne) was giving scientific first and last names to
plants and animals over 200 years ago. Ricinus is the Latin word for tick and is the
specific for the Mediterranean sheep tick (Ixodes ricinus). Apparently Linnaeus thought
the seeds looked like ticks, particularly large ticks engorged with blood (2,4,5)
.
Fig no.7: A western wood tick (Dermacentor occidentalis)
The mottled body of certain ticks superficially resembles a castor bean seed (especially
when the tick is engorged with blood) and the tick's head resembles the caruncle of a
castor bean seed.
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Fig no. 8 A] Ricinus communis plant B] Castor bean flowers & fruits
2.1.4 INTRODUCTION:
Ricinus communis Linn. is a tall glabrous and glaucous annual sometimes shrubby
or almost small tree, 2-4 m high; found throughout India, mostly growing wild on waste
land and also cultivated for its oil seeds.
Although it is native to the Ethiopian region of tropical east Africa, castor plant
has become naturalized in tropical and warm temperate regions throughout the world, and
is becoming an increasingly abundant weed in the southwestern United States. Castor
plants are very common along stream banks, river beds, bottom lands, and just about any
hot area where the soil is well drained and with sufficient nutrients and moisture to
sustain the vigorous growth. Although the seeds or beans are extremely poisonous, they
are the source of numerous economically important products and are one of earliest
commercial products. Castor beans have been found in ancient Egyptian tombs dating
back to 4000 B.C. and the oil was used thousands of years ago in wick lamps for lighting.
To many people the castor plant is just an overgrown, undesirable weed, and yet it
produces one of nature's finest natural oils(5).
2.1.5 GEOGRAPHICAL DISTRIBUTION:
Probably native to Africa, Castor bean has been introduced and is cultivated in
many tropical and subtropical areas of the world, frequently appearing spontaneously(6). It
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is a native of N. E. tropical Africa. It is found throughout India, cultivated and found wild
up to 2400 meters(7). Castor Bean is originally native to northeastern Africa and the
Middle East. It has escaped cultivation and become naturalized as a weed almost
everywhere in the world that has a tropical or subtropical climate(8).
2.1.6 CULTIVATION:
A) GERMPLASM:
Reported from the African Center of Diversity, castor bean thereof is reported to
tolerate bacteria, disease, drought, fungi, high pH, heat, insects, low pH, nematodes, poor
soil, salt, slope, virus, weed, wind, and wilt. Many developed; two of the best commercial
ones are: 'Conner' and 'Kansas Common', which give from 51.3 to 55.6% oil(9).
B) ECOLOGY:
Ranging from cool temperate moist to wet through tropical desert to wet forest life
zones, castor bean is reported to tolerate annual precipitation of 2.0 to 42.9 dm annual
temperature of 7.0 to 27.8°C and pH of 4.5 to 8.3. Grows best where temperatures are
rather high throughout the season, but seed may fail to set if it is above 38°C for an
extended period. Plant requires 140–180 day growing season and is readily killed by
frost. Irrigated crops require 2–3.5 acre-feet of water to produce satisfactory yields. High
humidity contributes to the development of diseases. Plants do best on fertile, well-
drained soils which are neither alkaline nor saline. Sandy and clay loam being best(10)
.
C) HARVESTING:
Fruits are harvested when fully mature and the leaves are dry, in about 95–180
days. In tropics, harvest is from wild or native plants. Planting and harvesting may be
done by hand methods or completely mechanized. Harvesting should begin before rainy
season in tropical regions, but in dry regions it is best to harvest when all fruits are
mature. In India fruit is picked in November; unless the capsules are dry, they must be
spread out to dry quickly. Castor oil is manufactured by running cleaned seed through the
decorticating machines to remove the seed coat from the kernel; the more complete this
operation the lighter the oil. Castor seeds cannot be ground or tempered as soybeans.
Unbroken or uncrushed seeds should be gotten to the press. Preheating may make heavy
viscous oil more mobile. Seed is put in 'cage' press, and number 1 oil is obtained, which
needs little refining but has to be bleached. Oil remaining in the press-cake is extracted by
solvent methods and is called number III oil, which contains impurities, and cannot be
effectively refined. Castor bean oil can be stored 3–4 years without deterioration(10)
.
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2.1.7 MORPHOLOGY:
A) Description of leaves:
Leaves green or reddish-green, broad, palmately lobed, with 5-11 lobes, 30-60 cm.
dia., nearly orbicular, lobes oblong linear, acute or acuminate, margin serrate, vary from
4-20 cm in length, 2.5 -7.5 cm in width; petiole 10-20 cm long, cylindrical or slightly
flattened towards distal and palmately attached to the blade, solid when young, becomes
hollow on maturity smooth, alternate, palmately-divided and 20-60 cm in width. The
lobes are oblong and toothed (11)
.
B) Castor bean flowers & fruits:
Flowers occur most of the year in dense terminal clusters (inflorescences), with
female flowers just above the male flowers. This species is clearly monoecious, with
separate male and female flowers on the same individual. There are no petals and each
female flower consists of a little spiny ovary (which develops into the fruit or seed
capsule) and a bright red structure with feathery branches (stigma lobes) that receives
pollen from male flowers. Each male flower consists of a cluster of many stamens which
literally smoke as they shed pollen in a gust of wind.
C) Castor seeds:
The shiny seeds of castor plants are a little larger than pinto beans and have very
beautiful and intricate designs. At one end is a small, spongy structure called the caruncle,
which aids in the absorption of water, when the seeds are planted. Like human faces,
finger prints or the spots on a leopard, no two seeds have exactly the same pattern. They
are unquestionably among the most deadly seeds on earth, and it is their irresistible
appearance that makes them so dangerous. The many "faces" of castor seeds like the faces
and fingerprints of the people. The beautiful designs on castor seeds exhibit infinite
genetic variation. The small structure on the end of each seed is a caruncle (4).
2.1.8 PHYSICAL CONSTITUENTS:
Table no.11: Physical constituents in 100 g of leaves of Ricinus communis Linn.
Total ash 12.4 g Fat 5.4 g
Fiber 24.8 g Total carbohydrate 57.4 g
Protein 10.3 g
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2.1.9 PHYTOCHEMISTRY:
Per 100 g, the leaves are reported to contain on a zero-moisture basis, 2,670 mg
calcium, and 460 mg phosphorous. The leaves contain isoquercetin 2, 5-dihydroxy
benzoic acid and epicatechin. They also contain rutin, hyperoside, quercetin, chlorogenic
acid, neochlorogenic acid and gallic acid. They contain an invertase, a glycoprotein
activated by macromolecules including proteins and lectins from castor (10)
.
The seed contains 5.1–5.6% moisture, 12.0–16.0% protein, 45.0–50.6% oil, and
2.0–2.2% ash. Seeds are high in phosphorus, 90% in the phytic form. The bean coat
yielded lupeol and 30-norlupan-3-ol-20-one. Seed coat contained 62% lipids and higher
amounts of phosphatides and non-saponifiable matter than seed kernel. The seeds contain
a powerful lipase, employed for commercial hydrolysis of fats, also amylase, invertase,
maltase, endotrypsin, glycolic acid, oxidase, ribonuclease, and a fat-soluble zymogen.
Sprouting seeds contain catalase, peroxidase and reductase, ricinus lipase, fixed oil, 49-
85% ricin, ricinin.
Roots, stems and leaves contain several amino acids. Flowers gave apigenin,
chlorogenin, rutin, coumarin and hyperoside. Castor oil is constituted by several fatty
acids.
The castor oil consists principally of ricinoleic acid with only small amounts of
dihydroxystearic, linoleic, oleic, and stearic acids. The unsaponifiable matter contains
sitosterol(12)
.
A) Chemistry of plant oils:
Plant oils are typically composed of triglyceride molecules (technically called
esters) composed of a 3-carbon alcohol (glycerol) plus three 18-carbon (or 16-carbon)
fatty acids. Unlike the saturated fatty acids of animal fats which are solid at room
temperature, plant fatty acids are typically unsaturated and liquid at room temperature,
with one or more double bonds between the carbon atoms (mono-unsaturated and
polyunsaturated)(13)
.
PARTS UTILISED: Roots, leaves, seeds.
PROPERTIES:
The root is sweetish, heating, plain tasting, neutral-natured; leaves and stems-
sweet-pungent tasting, neutral natured, slightly toxic. Seeds are exceedingly pungent in
taste, warming-natured. Soothes and regulates the gastrointestinal tract.
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2.1.10 FOLKLORIC USES:
It used in treatment of rheumatic arthritis, paralysis; epilepsy; distention of the
uterus, prolapsus drink dried root decoction or poultice Bai-hui pt (GV-20) with pounded
seed or leaf material. Used in non-lowering of the fetus (during delivery): poultice
Yungchuan Pt (K-1 pt) with pounded fresh leaves. In facial paralysis: poultice with
pounded seeds (seed coat removed). If the paralytic side is on the left side of the face,
apply poultice on the left. In wound caused by piercing with pointed objects (nails,
bamboo slats, bullet wound): use pounded fresh seed and apply as poultice. In skin ulcers:
Boil pounded leaves use as wash. Bark of castor plant also used as dressing for ulcers and
sores. Seed oil is laxative and vermicide; also used as ear drops to hardened cerumen and
also used for warts. For Milk stimulation: Pound leaves apply over breast as poultice. In
hemorrhoids, Roast seed, pound apply to affected area(14)
.
It is considered anodyne, antidote, bactericide, cathartic, cyanogenetic, discutient,
emetic, emollient, expectorant, insecticide, lactagogue, larvicidal, laxative, poison,
purgative, tonic, and vermifuge. Castor or castor oil is a dangerous ingredient in folk
remedies for abscess, arthritis, asthma, boils, burns, cancer, carbuncles, catarrh, chancre,
cholera, cold, colic, convulsions, corns, deafness, delirium, dermatitis, dogbite, dropsy,
epilepsy, fever, flu, gout, guineaworm, headache, inflammation, moles, myalgia, nerves,
osteomyelitis, parturition, rash, rheumatism, scrofula, seborrhea, skin, sores,
stomachache, swellings, toothaches, tuberculosis, tumors, urethritis, uteritis, venereal
disease, warts and wounds. Castor-oil is a cathartic and has labor-inducing properties.
Ricinoleic acid has served in contraceptive jellies. Ricin, a toxic protein in the seeds, acts
as a blood coagulant. Oil used externally for dermatitis and eye ailments. Seeds, which
yield 45–50% of a fixed oil, also contain the alkaloids ricinine and considered purgative,
counter-irritant in scorpion-sting and fish poison. Leaves applied to head to relieve
headache and as a poultice for boils(13,14,15)
.
A) USES OF CASTOR IN AYURVEDA.
Castor is a perennial evergreen shrub. The Sanskrit name erandah describes the
property of the drug to dispel diseases. It is considered as a reputed remedy for all kinds
of rheumatic affections. They are useful in gastropathy such as gulma, amadosa,
constipation, inflammations, fever, ascitis, bronchitis, cough, leprosy, skin diseases, and
vitiated conditions of vata, colic and lumbago.
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Root: The root is sweetish, heating, carminative; useful in inflammations, pains, asicites,
fever, glands, asthma, eructations, bronchitis, leprosy, diseases of the rectum, and the
head.
Leaves: The leaves are useful in “vata” and “kapha”, intestinal worms, night blindness,
earache; increases biliousness. Leaves are useful in burns, nyctalopia, strangury and for
bathing and fermentation and vitiated conditions of vata, especially in rheumatoid
arthritis and arthralgia. Fresh leaves are used by nursing mothers in the Canary Island as
an external application to increase the flow of milk.
Flowers: The flowers are useful in glandular tumors, anal troubles, vaginal pain. Flowers
are useful in arthralgia.
Fruit: The fruit is heating and an appetizer useful in tumors and pain, “vata”, piles,
diseases of the liver and spleen.
Seed: The seed is cathartic and aphrodisiac. The oil is sweetish; cathartic, aphrodisiac,
anthaelmintic, alternative; useful in tumors, diseases of the heart, slow fevers,
inflammations, asicites, typhoid, pain in the back, lumbago, leprosy, elephantiasis,
convulsions; increases “kapha” causes biliousness. Seeds are useful in dyspepsia and for
preparing a poultice to treat arthralgia.
Castor oil: Castor oil is an excellent solvent of pure alkaloids and as such solutions of
atropine, cocaine, etc. are used in ophthalmic surgery. It is also dropped into the eye to
remove the after-irritation caused by the removal of foreign bodies. The oil from seeds is
a very effective purgative for all ailments caused by vata and kapha. It is also
recommended for ascites, intermittent fever, colonitis, lumbago, coxalgia and coxitis.
Castor oil is also used for soap making.
B) USES OF CASTOR IN YUNANI:
The root bark is purgative, alternative; good in skin diseases. The leaves are
galactogogue; good for burns, the seeds and the oil from them have a bad taste; purgative;
useful in liver troubles, pain in the body, lumbago, boils, piles, ringworm, paralysis,
inflammations, ascites, asthma, rheumatism, dropsy and amenorrhoea(5,13)
.
C) MEDICINAL USES OF CASTOR SEEDS:
A poultice of castor seeds can be applied with gratifying results to gouty and
rheumatic swellings. A decoction of the roots of castor plant with carbonate of potash is
useful in the treatment of lumbago, rheumatism and sciatica. A paste of the kernel without
the embryo, boiled in milk, is also given as a medicine in these conditions. Castor oil is a
harmless purgative. It simply passes out after completing its purgative action, making the
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patient feel a mild irritation in the anus at that time. Administering of castor oil as a
purgative is very simple. About 30 to 60 grams of pure odorless castor oil is given orally
with 250 to 375 grams of lukewarm milk. It acts just after an hour. Those who find its use
nauseating and unpalatable can take it with ginger water or aqua anisi in place on milk.
This greatly reduces its unpleasantness, while destroying mucous and promoting healthy
appetite.
A poultice of castor leaves is useful as an external application of boils and
swellings. Coated with some bland oil such as coconut oil and heated, the hot leaves can
be applied over guinea-worm sores to extract the worms. A poultice of castor seeds is also
applied to scrofulous sores and boils due to tuberculosis of lymph nodes. Castor oil
massaged over the breast after child-birth increases the flow of milk, as it stimulates the
mammary glands. The leaves of castor can also be used to foment the breast for the same
purpose. Castor oil massaged over the body, before bath, keeps the skin healthy and
imparts sound sleep. Such an oil bath may be taken once in a week. Applying castor oil
over hand and feet before going to bed keeps them soft and similarly over the eyebrows
and eyelashes keeps them well-groomed. Used regularly as hair oil, it helps for growth of
the hair and cure dandruff(16)
.
2.1.11 PHARMACOLOGICAL ACTIVITY:
1] Anti-inflammatory activity:
The effect of petroleum ether extract of root of Ricinus Communis (150 mg/kg
p.o) has been investigated against Carrageenan, 5-Hydroxytryptamin, Dextran,
Bradykinin and Prostaglandin E, induced rat’s hind paw oedcma. The extract exhibited
significant anti-inflammatory activity against all the phlogestic agents except PGE. Mean
changes in the paw volumes revealed, the efficacy of extract against the phlogestic agents
in the following order: Carrageenan>Bradykinin>5-HT>Dextran. The anti-inflammatory
activity was compared with standard drugs such as Phenylbutazone and Betamethasone,
both in acute and chronic experimental models of inflammation in albino rats(17)
.
2] Antidiabetic activity:
Administration of the effective dose of Ricinus communis to the diabetic rats for
20 days showed favorable effects not only on fasting blood glucose, but also on total lipid
profile and liver and kidney functions on 10th and 20
th day. Ricinus communis seemed to
have a high margin of safety as no mortality and no statistically significant difference in
alkaline phosphatase, serum bilirubin, creatinine, serum glutamate oxaloacetate
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transaminase, serum glutamate pyruvate transaminase and total protein was observed
even after the administration of the extract at a dose of 10 g/kg body weight. Thus Ricinus
communis seems to have a promising value for the development of a potent
phytomedicine for the diabetes(18)
.
3] Anti-inflammatory and free radical scavenging activity:
Anti-inflammatory and free radical scavenging activities of the methanolic extract
of Ricinus communis Linn. root was studied in wistar albino rats. The methanolic extract
at doses 250 and 500 mg/kg p.o. exhibited significant anti-inflammatory activity in
carrageenan-induced hind paw edema model. The extract at the dose of 500 mg/kg p.o.
also exhibited significant (P < 0.001) anti-inflammatory activity in cotton pellet
granuloma model. The methanolic extract showed significant free radical scavenging
activity by inhibiting lipid peroxidation initiated by carbon tetrachloride and ferrous
sulphate in rat liver and kidney homogenates. The extract enhanced free radical
scavenging activity of stable radical 2,2-diphenyl-1-picryl-hydrazyl (DPPH radical hot),
nitric oxide and hydroxyl radical in vitro assay methods. The methanolic extract of
Ricinus communis Linn. root possess significant anti-inflammatory activity in acute and
chronic inflammatory models in rats(19)
.
4] Gastric Ulcer and Secretion activity:
Gastric ulcer and secretion studies in Pylorus ligated rats was conducted in albino
rats of either sex by administering Ricinus communis. The gastric juice was estimated for
its volume, acid output and peptic activity. Ulcer index was calculated by adding the
number of ulcers/erosions (in glandular portions of the stomach) with severity in pluses
(+) scored as l-4 per stomach after histological confirmation(19)
.
5] Anticancer activity (Ricin)
Although it is a very potent poison, ricin has been shown to possess antitumor
qualities and has been used in cancer research and chemotherapy during recent years. One
of the most promising uses of ricin is in the production of immunotoxins, where the
protein ricin is joined to monoclonal antibodies. The antibodies are produced in a test
tube (in vitro) and have protein receptor sites that recognize the specific target cells of a
tumor. The resulting ricin-antibody conjugate is called an immunotoxin. By arming these
antibodies with ricin, the deadly toxin can be carried directly to the site of the tumor in a
cancer patient. Thus, ricin can destroy the tumor cells, without damaging other cells in the
patient(20)
.
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6] Hepatoprotective activity:
Fresh leaves protected against liver injury induced by carbon tetra chloride in rats
while cold aqueous extract provided partial protection.
7] Purgative activity:
Castor oil was one of the old-fashioned remedies for everything from constipation
to heartburn widely used for over 2,000 years and is still used to this day; is the most
valuable laxative in medicines. It is considered to be fast, safe and gentle, prompting a
bowel movement in 3 - 5 hours, affecting the entire length of the bowel, but not
increasing the flow of bile, except in very large doses. The mode of its action is unknown.
It is recommended for both the very young and the aged. It is also used to clear the
digestive tract in cases of poisoning. It should not be used in cases of chronic
constipation.
8] Lubricant activity:
Castor oil is also used as lubricant. It is sometimes applied externally as a
soothing emollient for dry skin, dermatitis, other skin diseases, sunburn, open sores and it
is the primary ingredient of several brand name medications. Several additional little-
known uses for castor oil include hair tonics, ointments, cosmetics and contraception
creams and jellies(4).
2.1.12 BIOLOGICAL ACTIVITY:
The leaves (aqueous extract) exhibited 100% ovicidal and larvicidal activity
against mosquito larvae but have no effect on adult mosquitoes, thus used as larvicidal
agents in an integrated vector control. Castor leaves are used in preparing long acting
biocide compounds which are safe and stable and can completely control pests such as
mosquitoes, flies, cockroaches, ants, fleas and lice in 24 hours. The leaves have
insecticidal properties.
2.1.13 OTHER USES:
• The seed contains 35 - 55% of a drying oil. As well as being used in cooking, it is
an ingredient of soaps, polishes, flypapers, paints and varnishes.
• It is also used as a lubricant and for lighting and as an ingredient in fuels for
precision engines.
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• The oil is used in coating fabrics and other protective coverings, in the
manufacture of high-grade lubricants, transparent typewriter and printing inks.
• In textile dyeing (when converted into sulfonated castor oil or turkey-red oil, for
dyeing cotton fabrics with alizarine) and in the production of 'Rilson', a polyamide
nylon-type fibre.
• The dehydrated oil is an excellent drying agent which compares favorably with
tung oil and is used in paints and varnishes.
• The hydrogenated oil is utilized in the manufacture of waxes, polishes, carbon
paper, candles and crayons.
• A fiber for making ropes is obtained from the stems. Cellulose from the stems is
used for making cardboard, paper etc.
• The growing plant is said to repel flies and mosquitoes. When grown in the garden
it is said to rid it of moles and nibbling insects. The leaves have insecticidal
properties(11)
.
• Polyoxyethylene hydrogenated castor oil is used for preparing stable vitamin A
and E solubilised eye drops and oral nitroglycerine sprays.
• Polyoxyethylated castor oil is used as a surfactant for transdermal delivery of anti-
inflammatory drugs.
• Polyoxyethylated hydrogenated castor oil used to give finish to synthetic fiber to
provide high strength and low shrinkage(10)
.
2.1.14 TOXICITY
The seeds contain 2.8–3% toxic substances, 2.5–20 seed killing a man, 4 rabbits, 5
sheeps, 6 oxes, 6 horses, 7 pigs, 11 dogs, but 80 for cocks and ducks. The principal toxin
is the albumin, ricin. However, it produces antigenic or immunizing activity, producing in
small doses an antitoxin analogous to that produced against bacteria.
The seeds from the Ricinus communis Linn. are poisonous to people, animals and
insects. One of the main toxic proteins is "ricin", named by Stillmark in 1888 when he
tested the beans extract on red blood cells. If the seed is swallowed without chewing and
there is no damage to the seed coat, it will most likely pass harmlessly through the
digestive tract. However, if it is chewed or broken and then swallowed, the ricin toxin
will be absorbed by the intestines. It is said that just one seed can kill a child. Children are
more sensitive than adults to fluid loss due to vomiting and diarrhea, and can quickly
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become severely dehydrated and die. Perhaps just one milligram of ricin can kill an adult.
The symptoms of human poisoning begin within a few hours of ingestion and they are
abdominal pain, vomiting, diarrhea, sometimes bloody. Within several days severe
dehydration, a decrease in urine and a decrease in blood pressure occurs(1).
2.1.15 PRECAUTIONS
Repeated use of castor oil as a laxative should be avoided as it causes secondary
constipation, that is, recurrence of the condition after cure. Persons suffering from kidney
infections should not take castor oil as a purgative. It should also not be used when there
is abdominal pain or intestinal infections such as appendicitis, enteritis or inflammation of
the small intestine and peritonitis. Large doses of castor oil during the early months of
pregnancy may cause abortion(17)
.
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13. Duke JA and Wain KK. Medicinal Plants of the World. 1981.
14. http://www.stuartxchange.org/TanganTangan.html accessed on Aug 2008.
15. http://en.wikipedia.org/wiki/Castor_oil accessed on July 2008.
16. http://www.home-remedies- guide.com/herbs/castor-seeds.htm accessed on Aug
2008.
17. Banerjee S, Mukherjee A, Bandyqpadhyay SK, Mukherjee PK and Sikdar S,
Preliminary ‘studies on the anti-inflammatory effects of Ricinus communis,
Indian Journal of pharmacology. 1990;22: 239-244.
18. Shokeen P, Anand P. Krishna M. and Tando V, Antidiabetic activity of 50%
ethanolic extract of Ricinus communis and its purified fractions. Food and
chemical Toxicology. 2008; 46(11): 3458-3466.
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19. Ilavarasan R, Mallika M and Subramanian V, Anti-inflammatory and free radical
scavenging activity of Ricinus communis root extract, Journal of
Ethanopharmacology. 2006; 103(3): 478-480.
20. Banerjee S, Bandyopadhyay SK, Mukherjee PK, Mukherjee A and Sikdar S.
Further studies on the anti – inflammatory activities of Ricinus communis in
albino rat. Indian Journal of pharmacology.1991; 23: 149-152.
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2.2 PLANT PROFILE OF VITEX NEGUNDO LINN.
Uniyal et al. reiterates a popular local quote of the Bangalis in the Western
Himalayan region of India which translates as – A man cannot die of disease in an area
where Vitex negundo, Adhatoda vasica and Acorus calamus are found (provided that he
knows how to use them). The plant holds great promise as a commonly available
medicinal plant and it is indeed no surprise that the plant is referred to in the Indian
traditional circles as ‘sarvaroganivarini’ – the remedy for all diseases(1).
2.2.1 TAXONOMICAL CLASSIFICATION:
Kingdom: Plantae- Plants
Subkingdom: Tracheobionta – Vascular plants
Super division: Spermatophyte – Seed plants
Division: Magnoliophyta – Flowering plants
Class: Magnoliopsida – Dicotyledons
Subclass: Asteridae
Order: Lamiales
Family: Verbenaceae – Verbena family
Genus: Vitex Linn.
Species: Vitex negundo Linn. – (Chastetree)(2,3)
2.2.2 SYNONYMS:
Table no.12: Data showing synonyms of Vitex negundo Linn. in different languages
Sanskrit Indrani, Nilanirgundi, Nilapushpa, Nirgundi, Nirgundika, Renuka,
Sephalika, Shephali, Shvetasurasa, Sindhooka, Sindhuvaram.
Hindi Mewri, Nengar, Ningori, Nirgandi, Nirgunda, Nisinda,
Panikisambhalu, Sambhal, Sambhalu, Nirgundi, Shimalu.
Urdu Sambhalu, Tukhm sambhalu.
Bengali Nisinda, Sinduari, Beguna, Nishinda, Nishinde.
Kannada Bile-nekki, Bilenekki, Karilakki, Lakkagida, Lakki, Lakki-gida,
Lakkili.
Malyalam Bem-nosi, Indrani, Karunocci, Noch-chi, Nochi, Vella-noch-chi.
Marathi Nirgunda, Nengar, Nirgur, Lingur, Nirguda, Nirgundi, Limgud,
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Negumd.
Oriya Thingkhawilupa, Niligundi.
Tamil Nallanocci, Nirkkundi, Nirkundi, Nochi, Sinduvaram,
Tiriburamerittan.
Telgu Nalla-vavili, Nallavavili, Sindhuvaruma, Sinduvaramu, Tell-vavili.
Fig. no. 9: Vitex negundo Linn. plant(4)
2.2.3 INTRODUCTION:
Nirgundi has been used since ancient times as a female remedy and also for pains
in Ayurveda and also in Roman medicine. One of its properties was to reduce sexual
desire, and it is recorded that Roman wives whose husbands were abroad with the legions
spread the aromatic leaves on their couches for this purpose. It became known as the
chaste berry tree. During the middle ages, Chaste berry’s supposed effect on sexual desire
led to it becoming a food spice at monasteries, where it was called "Monk's pepper" or
"Cloister pepper". Hippocrates, Dioscorides, and Theophrastus mention the use of Vitex
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for a wide variety of conditions, including hemorrhage following childbirth, and also to
assist with the “passing of afterbirth”(5,6)
.
2.2.4 GEOGRAPHICAL DISTRIBUTION:
Vitex usually grows from three to nine feet tall, but under cultivation can develop
to 20 feet tall. Nirgundi occur in tropical to temperate regions (up to 2200 m from east to
west) grows gregariously in wastelands and is also widely used as a hedge-plant. This
species is globally distributed in Indo-Malesia, cultivated in America, Europe, Asia and
West Indies. Within India, it is found throughout the greater part of India, ascending to an
altitude of 1500 metres in the outer Himalayas(7).
It is abundant in open-waste lands. It is widely planted as a hedge-plant along the
roads and between the fields. It is widely distributed throughout India, Sind, Ceylon,
Afghanistan, Philippine Islands and Tropical Africa, Madagascar and China. Locally
distributed throughout the State Maharashtra along the banks of rivers; very common near
the sea-coast in tidal and beach-forests in Konkan; along Deccan rivers. Habitat found to
be in Waste lands and moist situations(3).
Vitex negundo Linn is large and erect aromatic shrubs grow to height 2–5 m or
sometimes, a small slender tree with quadrangular branchlets densely whitish, tomentose
branchlets distributed throughout India. It is often found growing next to streams and it
loves water(6,8)
.
2.2.5 CULTIVATION:
It is widely planted as a hedge plant in between the fields and usually not browsed
by the cattle. It can be reproduced readily from shoot cuttings. It produces root suckers
which can also be utilized as planting material. An easily grown plant, it prefers a light
well-drained loamy soil in a warm sunny position sheltered from cold drying winds
succeeds in poor dry soils. Plants tolerate temperatures down to about -10°C. The leaves
and stems are strongly aromatic. The flowers have a most pronounced musk-like
perfume(9).
2.2.6 PHARMACOGNOSY:
Root: Roots are woody, fairly thick, 8-10 cm in diameter; external surface brownish,
rough due to the presence of longitudinal fissures and a small rootlets. The bark is very
thin and corky portion can be scrapped off easily. Transverse section shows outer cork
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consisting of 12-20 rows of nearly cubical to rectangular cells, the cells of peripheral
rows being thick walled but not lignified. Phellogen is single layered.
Stem Bark: Bark occurs in channelled pieces, 0.3-0.5 cm thick; outer surface yellowish
grey, rough, lenticelular, longitudinally channeled and transversely cracked; inner surface
darker than outer, blackish and smooth; fracture short and splintery; taste slightly bitter.
In transverse section the bark shows well developed periderm and secondary phloem
elements.
Leaf:
Morphology: Leaves are palmately compound, petiole 2.5-3.8 cm long; 3-5 foliate; the
middle leaflet is petiolate; in trifoliate leaf, leaflet lanceolate or narrowly lanceolate,
acute, entire or rarely crenate, middle leaflet 5-10 cm long and 1.6-3.2 cm broad, with 1-
1.3 cm long petiolule, remaining two sub-sessile; in pentafoliate leaf inner three leaflets
have petiolule and remaining two sub-sessile; Odour is agreeably aromatic surface
glabrous above and tomentose beneath; texture, leathery(10)
.
Microscopy: Petiole shows single layered epidermis having a number of unicellular,
bicellular and uniseriate multicellular covering trichomes and also glandular trichomes
with uni to tricellular stalk and uni to bicellular head; cortex composed of outer
collenchymatous tissue and inner 6-8 layers of parenchymatous tissue; collenchyma well
developed in basal region and gradually decreases in middle and apical regions; pericyclic
fibres absent in basal region of petiole and present in the form of a discontinuous ring in
apical region surrounding central horse shoe-shaped vascular bundle; a few smaller
vascular bundles present ventrally between arms of central vascular bundle and two, or
rarely three, bundles situated outside the arms.
Lamina - shows single layered epidermis having mostly unicellular hairs, bi and
multicellular and glandular trichomes being rare; hypodermis 1-3 layered interrupted at
places by 4-8 palisade layers containing chlorophyll; a large number of veins enclosed by
bundle sheath traverse mesophyll; stomata present only on the ventral surface, covered
densely with trichomes; vein-islet and vein termination number of leaf are 23-25 and 5-7
respectively.
Powder - shows number of pieces or whole, uni, bi and multicellular covering trichomes,
glandular trichomes, palisade tissues with hypodermis, and upper and lower epidermis,
xylem vessels with pitted walls(7).
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2.2.7 PHYSICAL CONSTITUENTS:
Table no.13: Quantitative (%) analysis of physical constituents(7, 11)
Total ash 6.055 % Carbohydrate 7.5-10.57 %
Acid insoluble ash 0.920 % Crude fiber 25.50-30.50 %
Water soluble ash 1.460 % Fat 5.00-9.00 %
Pet. Ether extractive 0.912 % Alkaloids 0.5 %
Benzene extractive 0.535 % Bitter 5 %
Chloroform extractive 0.433 % Proteins 12.22-15.23 %
Alcohol extractive 24.330 % Moisture 15.00-18.70 %
Water-soluble extractive Not less than 20 % Foreign Matter 1.9-2.0 %
2.2.8 PHYTOCHEMISTRY:
Table no.14: Phytochemical constituents of different plant parts of Vitex negundo
Linn.(5,7, 8,12,13,14,15,17)
PLANT PART PHYTOCHEMICAL CONSTITUENTS
Leaves
6′-p-hydroxybenzoyl mussaenosidic acid; 2′-p-hydroxybenzoyl
mussaenosidic acid viridiflorol; β-caryophyllene; sabinene; 4-
terpineol; gamma-terpinene; caryophyllene oxide; 1-oceten-3-ol;
globulol; 5,3′-dihydroxy-7,8,4′-trimethoxyflavanone; 5,3′-
dihydroxy,6,7,4′trimethoxyflavanone; 5-hydroxy-3,6,7,3',4'-
pentamethoxy flavones; 5,7dihydroxy-6,4' dimethoxy flavonone;
5hydroxy-7,4' dimethoxy flavones; 5,3'-dihydroxy-7,8,4'-trimethoxy
flavanone; betulinic acid [3β-hydroxylup-20-(29)-en-28-oic acid];
ursolic acid [2β -hydroxyurs-12-en-28-oic acid]; n-hentriacontanol; β-
sitosterol; p-hydroxybenzoic acid; protocatechuic acid; oleanolic acid;
flavonoids angusid; casticin; vitamin-C; nishindine; gluco-nonitol;
Seeds
3β-acetoxyolean-12-en-27-oic acid; 2α, 3α-dihydroxyoleana-5,12-
dien-28-oic acid; 2β,3α diacetoxyoleana-5,12-dien-28-oic acid; 2α, 3β-
diacetoxy-18-hydroxyoleana-5,12-dien-28-oic acid; vitedoin-A;
vitedoin-B; a phenylnaphthalene-type lignan alkaloid, vitedoamine-A;
five other lignan derivatives, 6-hydroxy-4-(4-hydroxy-3-methoxy-
phenyl)-3-hydroxymethyl-7-methoxy-3,4-dihydro-2-naphthaldehyde,
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β-sitosterol; p-hydroxybenzoic acid; 5-oxyisophthalic acid; n-
tritriacontane, n-hentriacontane; n-pentatriacontane; n-nonacosane.
Roots
2β,3α-diacetoxyoleana-5,12-dien-28-oic acid; 2α,3α-dihydroxyoleana-
5,12-dien-28-oic acid; 2α,3β-diacetoxy-18-hydroxyoleana-5,12-dien-
28-oic acid; vitexin; isovitexin, negundin-A; negundin-B; (+)-
diasyringaresinol; (+)-lyoniresinol; vitrofolal-E; vitrofolal-F, acetyl
oleanolic acid; sitosterol; 3-formyl-4.5-dimethyl-8- oxo-5H-6,7-
dihydronaphtho (2,3-b)furan.
Essential oil of
fresh leaves,
flowers and
dried fruits
δ-guaiene; guaia-3,7-dienecaryophyllene epoxide; ethyl-
hexadecenoate; α-selinene; germacren-4-ol; caryophyllene epoxide;
(E)-nerolidol; β-selinene; α-cedrene; germacrene D; hexadecanoic
acid; p-cymene and valencene. viridiflorol (19.55%), β-caryophyllene
(16.59%), sabinene (12.07%), 4-terpineol (9.65%), γ-terpinene
(2.21%), caryophyllene oxide (1.75%), 1-oceten-3-ol (1.59%), and
globulol (1.05%). Viridiflorol
PARTS USED: Whole plant
2.2.9 AYURVEDIC PROPERTIES:
Table no.15: Data showing Ayurvedic properties of Vitex negundo Linn.
Rasa Katu (pungent), Tikta (bitter)
Guna Laghu (Ruksha), Ruksha (dry)
Virya Ushna (hot)
Vipaka Katu (pungent)
Doshakarma Kapha-Vata Shamaka
Formulations (Yog):
1. Arkadi kvath churna
2. Manikya rasa
3. Vatavidhvamsana rasa
4. Maha vishgarbha tiala
5. Vishagarbh taila(1).
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Home remedies:
1. In cold, its decoction 20 ml should be used along with 1gm Pippali and 250 mg
Vacha.
2. In pneumonitis, Swarasa of its leaves 10 ml is so beneficial along with Pippali.
3. It’s paste on affected site is painkiller and anti-inflammatory(6).
2.2.10 MEDICINAL IMPORTANCE:
Herbal medicine, rather than merely curing a particular disease, aims at returning
the body back to its natural state of health. The phytochemical components of medicinal
plants often act individually, additively or synergistically in improvement of health. After
having analyzed the various chemical components present in different parts of Vitex
negundo Linn., it is imperative that focus shifts to the medicinal applications of the plant.
Myriad medicinal properties have been ascribed to Vitex negundo Linn. and the plant has
also been extensively used in treatment of a plethora of ailments. These properties have
been categorized under three heads – traditional medicine, folk medicine and
pharmacological evidence.
Leaves: The leaves of Vitex negundo Linn. are antibacterial, antitumor, astringent,
febrifuge, sedative, tonic and vermifuge. They are useful in dispersing swellings of the
joints from acute rheumatism and of the testes from suppressed gonorrhea. The juice of
the leaves is used for removing foetid discharges and worms from ulcers, whilst oil
prepared with the leaf juice is applied to sinuses and scrofulous sores. Extracts of the
leaves have shown bactericidal and antitumor activity. Leaves are antiparasitical,
alterative, aromatic, vermifuge, pain reliever. Leaves are insect repellents. Extracts of the
leaves have insecticidal activity. The fresh leaves are burnt with grass as a fumigant
against mosquitoes. Decoction of leaves may improve eyesight.
Dosage: Nirgundi Juice - 20 to 30 ml per day.
Nirgundi leaf Powder - 3 to 6 grams per day.
Stem: A decoction of the stems of Vitex negundo Linn. is used in the treatment of burns
and scalds.
Fruit: The dried fruit of Vitex negundo Linn. is vermifuge. The fruit is also used in the
treatment of angina, colds, coughs, rheumatic difficulties etc. The fresh berries are
pounded to a pulp and used in the form of a tincture for the relief of paralysis, pains in the
limbs, weakness etc. Fruit-nervine, cephalic, emengogue, dried fruit-vermifuge
employing an aqueous extract from the fruit, a 1979 study reported good results on
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premenstrual water retention. Women were able to sustain a good level of milk
production for breast feeding while taking this herb. While it took some time for the drug
to take effect, the women were able to continue the use of the drug for months without
harmful side effects.
Root: The root of Vitex negundo Linn. is expectorant, febrifuge and tonic. It is used in the
treatment of colds and rheumatic ailments. It is harvested in late summer and autumn and
dried for later use. Roots are tonic, febrifuge, expectorant, diuretic. Root juice is said to
increases the growth of hair(18)
.
Seed: Seeds of Vitex negundo Linn. occasionally used as a condiment, it has pepper
substitute. When washed to remove the bitterness it can be ground into a powder and used
as a flour, though it is very much a famine food used only when all else fails(8,9)
.
A) Traditional medicine
Traditional medicine mainly comprises of Indian Ayurveda, Arabic Unani
medicine and traditional Chinese medicine. In Asia and Latin America, populations
continue to use traditional medicine as a result of historical circumstances and cultural
beliefs. Traditional medicine accounts for around 40% of all health care delivered in
China. Up to 80 % of the population in Africa uses traditional medicine to help meet their
health care needs.
B) Ayurveda
The plant finds mention in the verses of the Charaka Samhita which is unarguably
the most ancient and authoritative textbook of Indian Ayurveda. Vitex negundo Linn. has
been designated as an anthelmintic and is prescribed as a vermifuge in the exposition on
the Charaka Samhita.
Other Ayurvedic uses of Vitex negundo Linn. are people sleep on pillows stuffed
with Vitex negundo Linn. leaves to dispel catarrh and headache and smoke the leaves for
relief. Crushed leaf poultice is applied to cure headaches, neck gland sores, tubercular
neck swellings and sinusitis. Essential oil of the leaves is also effective in treatment of
venereal diseases and other syphilitic skin disorders. A leaf decoction with Piper nigrum
is used in catarrhal fever with heaviness of head and dull hearing. A tincture of the root-
bark provides relief from irritability of bladder, rheumatism and in dysmenorrhea.
Formulations described in Anubhoga Vaidya Bhaga, a compendium of formulations in
cosmetology, in outlining the use of Vitex negundo Linn. leaves along with those of
Azadirachta indica, Eclipta alba, Sphaeranthus indicus and Carum copticum in a notable
rejuvenation treatment known as Kayakalpa.
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C) Unani medicine:
Vitex negundo Linn. is commonly known as Nisinda in Unani medicine. The seeds
are administered internally with sugarcane vinegar for removal of swellings. Powdered
seeds are used in spermatorrhoea and serve as an aphrodisiac when dispensed along with
dry Zingiber officinale and milk.
D) Chinese medicine
The Chinese Pharmacopoeia prescribes the fruit of Vitex negundo Linn. in the
treatment of reddened, painful and puffy eyes, headache and arthritic joints.
E) Uses in western herbal medicine
Modern medical world with Vitex began with the introduction of concentrate
extracts of Vitex fruits in the 1950. From 1943 to 1997, approximately 32 clinical trial
were conducted on a propriety Vitex agnus berry product for evaluating its effectiveness
in treating mastitis and fibrocystic diseases, menopausal symptoms, poor lactation,
uterine bleeding disorder and menstrual irregularities(18)
.
In homoeopathic medicine, Vitex agnus and Vitex negundo Linn. is used for
various sexual debilities- marked depression of vital power, premature old age with
apathy, self contempt for the sexual abuse nervous debility in unmarried person feeble
erection without sexual desire, emission of prolactic fluid when straining at stool, cold,
hard, swollen, painful testicle. In general practice, the drug is prescribes to female for
leukemia staining yellow suppressed menses, slangy or suppressed breast milk,
inflammation of uterus. The flowers are astringents and used in fever, diarrhea and liver
complaints. The fruits are prescribed in headache catarrh and watery eyes when dried. It
is consider vermifuge. They are much valued medicinally in china. An aqueous extract of
the fruits was found to be good analgesic action. In Philipins – the seed are reported to
eaten after boiling. The young shoots are used in the basket making. The ash of the plants
is source of potassium carbonate or peer ash and is reported to be used as an alkali in
drying(3)
.
F) Folk medicine
Folklore systems of medicine continue to serve a large segment of population,
especially those in rural and tribal areas, regardless of the advent of modern medicine.
The entries regarding the multifarious applications of Vitex negundo Linn. in folk
medicine have been grouped regionally to emphasize the ethanobotanical diversity and
ubiquity of the plant; and the details have been laid out in following tables.
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Table no. 16: Emphasizing the ethnobotanical diversity and ubiquity of the plant
Sr.
no. Country Region Local name Used in treatment of
1 Bangladesh Chittagong - - Weakness, Headache, Vomiting,
Malaria, Black fever
2 China Guangdong Buging′iab Common cold, Flu and Cough
3 Nepal Kali Gandaki Simali Sinusitis, Whooping cough
4 Pakistan
Buner Marvandaey Chest-pain, Backache, Used as
toothbrush
Kot
Manzaray
Baba valley
-- Used as anti-allergenic agent
Siran valley Kalgari Used as medicine for buffaloes in
colic
Margallah
hills Nirgud Gum and skin diseases
5 Philippines - - -- Cancer
6 Sri Lanka -- Nilnikka
Eye disease, Toothache,
Rheumatism
Used as a tonic, carminative and
vermifuge.
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Table no.17: Uses of Vitex negundo Linn. in folk medicine in India(12,13)
Sr.
no. State Region
Local
name Used in treatment of
1 Andhra
Pradesh Puttaparthi
Tella
Vaavili
Asthma, Cancer, Used as bath for
women in puerperal state and for
new born children
2 Assam -- Pochatia
Jaundice, Urticaria, Cellulitis,
Abcesses, Carbuncles, Eczema,
Liver disorders
3 Himachal
Pradesh
Garwahl
Parvati
valley
Sambhaalu
Bana
Kwashiorkor
Wounds, Body ache
4 Karnataka
Dharwad Lakki,
Karilakki Toothache
Mysore Bilenekki Febrile, catarrhal and rheumatic
afflictions
Uttara
Kanada Nekki Migraine
5 Maharashtra
Konkan Lingur Rheumatism
Amravati Samhalu Encephalitis
Chota
Nagpur Nirgundi Expectorant
Satpuda - Joint pain
6 Orissa Malkangiri Languni Jaundice
7 Tamil Nadu Southern
parts Notchi Used as antidote for snake bite
8 Uttar
Pradesh
Jaunsar-
Bawar hills Somi Eye pain
Moradabad Mala Used as refrigerant for cattle
Uttaranchal - 48 types of ailments
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2.2.11 PHARMACOLOGICAL EVIDENCE:
Demands of the scientific community have necessitated experimental evidence to
further underline the medicinal importance of Vitex negundo Linn. described above.
Taking cue from these traditional and folk systems of medicine, scientific studies have
been designed and conducted in order to pharmacologically validate these claims.
The decoction of leaves is used for treatment of inflammation, eye-disease,
toothache, leucoderma, enlargement of the spleen, ulcers, cancers, catarrhal fever,
rheumatoid arthritis, gonorrhea, sinuses, scrofulous sores, bronchitis and as tonics. As
vermifuge, lactagogue, antibacterial, antipyretic, antihistaminic, analgesic, insecticidal,
ovicidal, growth inhibition and morphogenetic agents. antigenotoxic, antihistamine, CNS
depressant activity and anti-fertility effects were reported from the leaves of Vitex
negundo Linn.(13)
.
1] Anti-inflammatory activity:
The sub-effective dose of Vitex negundo Linn. potentiated anti-inflammatory
activity of phenlbutazone and ibuprofen significantly in carrageenin induced hind paw
oedema and cotton pellet granuloma models. The potentiation of anti-inflammatory
activities phenlbutazone and ibuprofen by Vitex negundo Linn. indicates that it may be
useful as an adjuvant therapy along with standard anti-inflammatory drugs.
Yunos et al. and Jana et al. established anti-inflammatory properties of Vitex
negundo Linn. extracts in acute and sub-acute inflammation which are attributed to
prostaglandin synthesis inhibition(3,19)
.
2] Antinociceptive activity:
Tail flick test in rats and acetic acid induced writhing in mice were employed to
study the antinociceptive activity of ethanolic leaf extract of Vitex-negundo Linn. (100,
250 and 500 mg/kg, p.o). The effect was compared with meperidine (40 mg/kg, sc) in tail
flick method and aspirin (50 mg/kg, p.o) in writhing test as a standard control
respectively. An interaction with naloxone hydrochloride was also studied in tail flick
method for its mechanism of central analgesic action. It showed significant analgesic
activity in dose dependant manner in both the experimental models. It suggested that
Vitex-negundo Linn. possesses both central and peripheral analgesic activity. The central
analgesic action does not seem to be mediated through opioid receptors. It may prove to
be a useful adjuvant therapy along with standard analgesic drug(20)
.
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3] CNS depressant activity:
A methanolic extract of the leaves of Vitex negundo Linn. was found to significantly
potentiate the sleeping time induced by pentobarbitone sodium, diazepam and
chlorpromazine in mice(8).
4] Antifungal activity:
Bioactivity guided fractionation of ethanolic extract of leaves of Vitex negundo Linn.
resulted in the isolation of new flavone glycoside along with five known compounds. All
the isolated compounds were evaluated for their antimicrobial activities. The new flavone
glycoside and compound 5 were found to have significant antifungal activity against
Trichophyton mentagrophytes and Cryptococcus neoformans at MIC 6.25 µg/ml(21,22)
.
5] Antioxidant Activity:
The antioxidant potency of Vitex negundo Linn. was investigated by all the fractions
of Vitex negundo Linn. exhibited a potent scavenging activity for (2, 2′-azino-bis 3-ethyl
benzothiazoline-6-sulfuric acid) ABTS radical cations in a concentration dependent
manner, showing a direct role in trapping free radicals. The polar fractions of Vitex
negundo Linn. possess potent antioxidant properties. Tandon and Gupta have also
reported similar antioxidant properties of Vitex negundo Linn. in rats, by using ethanol
induced oxidative stress model(22,23)
.
The extracts also possess the ability to combat oxidative stress by reducing lipid
peroxidation owing to the presence of flavones, vitamin C and carotene. Rooban et al.
evaluated the antioxidant and therapeutic potential of Vitex negundo Linn. flavonoids in
modulating solenoid-induced cataract and found it to be effective(3,24)
.
6] Enzyme-inhibitory activity:
Root extracts of Vitex negundo Linn. showed inhibitory activity against enzymes such
as lipoxygenase and butyryl-cholinesterase, α-chymotrypsin, xanthine-oxidase and
tyrosinase. Also reported the HIV type 1 reverse transcriptase inhibitory activity of the
water extract of the aerial parts of Vitex negundo Linn. (25)
.
7] Anticonvulsant activity:
Maximal electroshock seizures (MES) in albino rats and pentylenetetarazole (PTZ)
induced seizures in albino mice were used to study anticonvulsant activity of Vitex
negundo Linn. leaf extract. The test drug dose (1000 mg/kg, p.o) showed 50% protection
in clonic seizures and 24-hour mortality against PTZ induced seizures. It also decreased
number and duration of convulsions significantly. Vitex negundo Linn. potentiated
anticonvulsant activity of valporic acid. The anticonvulsant activity of Vitex negundo
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Linn. has not been found equi-effective with standard drugs. Moreover, the potentiation
of diphenylhydantoin and valporic acid by Vitex negundo Linn. indicates that it may be
useful as an adjuvant therapy along with standard anticonvulsants and can possibly lower
the requirement of diphenylhydantoin and valporic acid(3,26)
.
8] Antibacterial studies:
Essential oils and successive ethyl acetate and ethanol extracts of Vitex negundo
Linn. showed antibacterial activity against Staphylococcus aureus, Bacillus subtilis,
Escherichia coli and Pseudomonas aeruginosa bacterial strains.
Main constituents identified in leaves oil were d-guaiene, carryophyllene epoxide
and ethylhexadecenoate; In flowers oil α-selinene, germacren-4-ol, carryophyllene
epoxide and (E)-nerolidol while fruit oil showed β -selinene, α-cedrene, germacrene D
and hexadecanoic acid as the main constituents which help for antibacterial activity.
9] Antiallergic Activity:
Ethanolic extract of Vitex negundo Linn. showed antiallergic activity against
immunologically induced degranulation of mast cells. It also inhibited edema during
active paw anaphylaxis in mice. The extract significantly inhibited both the initial and
later sustained phases of tracheal contractions. The initial phase was primarily due to
histamine and the latter phase was due to release of lipid mediators from arachidonic acid.
Inhibition of the latter phase may be secondary to inhibition of arachidonic acid by the
ethanolic extract.
10] Snake venom neutralization activity:
The methanolic root extracts of Vitex negundo Linn. and Emblica officinalis
showed antisnake venom activity. The plant Vitex negundo Linn. extracts significantly
antagonized the Vipera russellii and Naja kaouthia venom induced lethal activity both in
in vitro and in vivo studies. Vipera russellii venom-induced haemorrhage, coagulant,
defibrinogenating and inflammatory activity were significantly neutralized by both plant
extracts. No precipitating bands were observed between the plant extract and snake
venom(8,26)
.
11] Effect on reproductive potential:
The flavonoid rich fractions of seeds of Vitex negundo Linn. caused disruption of
the latter stages of spermatogenesis in dogs and interfered with male reproductive
function in rats. It must however be noted that these findings are in sharp contrast with
the traditional use of Vitex negundo Linn. as aphrodisiac. Hu et al. determined that
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ethanolic extracts of Vitex negundo Linn. showed estrogen-like activity and propounded
its use in hormone replacement therapy(8,27)
.
12] Histomorphological and cytotoxic effects:
Tandon and Gupta studied the histomorphological effect of Vitex negundo Linn.
extracts in rats and found the stomach tissue to be unaffected even by toxic doses; while
dose-dependent changes were observed in the heart, liver and lung tissues. Cytotoxic
effect of leaf extracts of Vitex negundo Linn. was tested and affirmed using COLO-320
tumour cells. On one hand, Diaz et al. found the chloroform extracts of Vitex negundo
leaves to be toxic to a human cancer cell line panel. Yunos et al. reported that Vitex
negundo Linn. extracts were non-cytotoxic on mammary and genito-urinary cells of
mice(28,29)
.
13] Hepatoprotective activity:
The ethanolic extract of Vitex negundo Linn. at 250 and 500 mg/kg doses
significantly decrease Serum Bilirubin, Aspartate Aminotransferase (AST), Alanine
Aminotransferase (ALT), Alkaline Phosphates (ALP) and Total Protein (TP) levels
against hepatotoxicity (HT) produced by administering a combination of three
antitubercular drugs isoniazide (7.5 mg/kg), rifampin (10 mg/kg) and pyrazinamide (35
mg/kg). Alcoholic extract of the seeds of Vitex negundo Linn. showed the
hepatoprotective action against carbon tetrachloride-induced liver damage. The extract
was found to be effective in preventing liver damage which was evident by
morphological, biochemical and functional parameters(3).
Nirgundi exerts a protective effect on CYP2E1-dependent CCl4 toxicity via
inhibition of lipid peroxidation, followed by an improved intracellular calcium
homeostasis and inhibition of Ca2+ dependent proteases
(30).
14] Hypoglycemic activity:
Villasenor and Lamadrid have provided an account of the anti-hyperglycemic
activity of Vitex negundo Linn. leaf extracts(31)
.
15] Laxative activity:
The aqueous extract of the Vitex negundo Linn. leaves at doses 100 and 200
mg/kg was investigated for laxative activity according to Cappaso et al. in albino rats
were compared with standard drug agar-agar (300 mg/kg, p.o.) in normal saline(32)
.
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16] Immunomodultory activity:
Immunomodulatory effect of Vitex negundo Linn. extracts has been reported by
Ravishankar and Shukla .The decoction of leaves is considered as tonic, vermifuge and is
given along with long pepper in catarrhal fever(32)
.
17] Drug potentiating ability:
Administration of Vitex negundo Linn. extracts potentiated the effect of
commonly used anti-inflammatory drugs such as ibuprofen and phenylbutazone
analgesics such as meperidine, aspirin, morphine and pethidine; sedative-hypnotic drugs
like pentobarbitone, diazepam and chlorpromazine; anti-convulsive agents such
diphenylhydantoin and valporic acid(23)
.
2.2.12 BIOLOGICAL ACTIVITY:
Vitex negundo Linn. has shown promise as an effective bio-control agent. The
extracts of Vitex negundo Linn. possess inhibitory, deterrent or lethal activity on
biological agents that cause disease and damage to other organisms.
Table no. 18: Data summarizes the effect of Vitex negundo Linn. on different
pathogens and pests
ACTIVITY ACTION AGAINST
Anti-bacterial Escherichia coli, Klebsiella aerogenes, Proteus vulgaris and
Pseudomonas aerogenes (Bacteria)
Anti-feedant Spodoptera litura (Asian army-worm), Achoea janata (Castor
semi-looper)
Anti-filarial Brugia malayi (Microfilarial parasite)
Anti-fungal
Alternaria alternata, Curvularia lunata Trichophyton
entagrophytes, Cryptococcus neoformans, Aspergillus niger,
Candida albicans
Anti-larval Cnaphalocrocis medinalis (Rice leaf-folder)
Anti-viral Plasmodium falciparum (Virus)
Insecticidal
Callosobruchus maculatus (Pulse beetle)
Phthorimaea operculella (Potato-tuber moth)
Sitotroga cerealella (Angoumois grain moth)
Aphis citricola (Spirea aphid), Aphis gossypii (Melon or Cotton
aphid), Myzus persicae (Green peach aphid)
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Larvicidal
Anopheles subpictus, Culex tritaeniorhynchus (Mosquitoes)
Culex quinquefasciatus (Mosquito)
Anopheles stephensi (Mosquito)
Plutella xylostella (Diamond-back moth)
Mosquito repellant Culex tritaeniorhynchus (Mosquito)
Mosquito repellant Aedes aegypti (Mosquito)
Additionally, production of plant secondary metabolites de novo, by in vitro cell
culture methods, has assumed importance in the last two decades because the structural
complexity of naturally occurring metabolites forms the basis for the chemical synthesis
of novel and more potent analogues.
2.2.13 CONTRAINDICATIONS:
Nirgundi should be used with caution with the concurrent use of psychotropic
drugs, including analgesics, sedatives, antidepressants, anticonvulsants and
antipsychotics. Vitex negundo Linn. is quite similar botanically to the better studied Vitex
agnus castus, and thus may have a similar range of contraindications, including the
concurrent use of progesterogenic drugs and hormone replacement therapies(8).
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REFERENCES:
1. http://www.ayurvedaconsultants.com/herb_consult.aspx?commonName=NIRGU
NDI accessed on Nov 2010.
2. Bansod MS, Harle UN, Vitex negundo L: Phytochemical constituents, traditional
ses and pharmacological properties: Comprehesive review. Pharmacologyonline
Newsletter. 2009; 1: 286-302.
3. Mahalakshmi R, Rajesh P, Ramesh N, Balsubramanian V, Kanan VR.
Hepatoprotective activity on Vitex negundo linn (verbanaceae) by using wistar
albino rats in ibuprofen induced model. International journal of pharmacology.
2010: 1-6.
4. http://www.blissayurveda.com/pics/Vitexnegundo.jpg accessed on Oct 2009.
5. http://planetayurveda.com/nirgundipowder.htm accessed on Aug 2008.
6. http://www.planetayurveda.com/home-remedies.htm accessed on Aug 2008.
7. The Ayurvedic Pharmacopeia of India. Government of India Ministry of health
and family welfare department of ISM & H. 2001; Part-I, Vol 3: 142-144.
8. http://www.toddcaldecott.com/index.php/herbs/learning-herbs/315-nirgundi
accessed on July 2009.
9. http://www.pfaf.org/index.php accessed on July 2009.
10. http://www.impgc.com/plantinfo_A.php?id=942&bc accessed on Oct 2010.
11. http:// Crude protein www.nirgundi protocol.htm accessed on Sept 2010.
12. Vishwanathan AS, Basavaraju R, A review on Vitex negundo L. – A medicinally
important plant. EJBS April-June 2010; 3 (1): 30-42
13. Gautam LN, Shrestha SL, Wagle P, Tamrakar BM, Chemical constituents from
vitex negundo (linn.) of nepalese origin Scientific World. 2008; 6 (6).
14. http://www.himalayahealthcare.com/aboutayurveda/cahv.htm accessed on Sept
2010.
15. Singh V, Dayal R, Bartley JP, Chemical constituents of Vitex negundo leaves.
Journal of Medicinal and Aromatic Plant Sciences. 2003; 25: 94-98.
16. Singh V, Dayal R, Bartley JP, Volatile Constituents of Vitex negundo Leaves.
Planta Med. Letters, 1999; 65: 580-582.
17. Meena AK, Singh U, Yadav AK, Singh B, Rao MM, Pharmacological and
Phytochemical Evidences for the Extracts from Plants of the Genus Vitex – A
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69
Review. International Journal of Pharmaceutical and Clinical Research. 2010;
2(1): 01-09
18. http://www.philippineherbalmedicine.org/lagundi.htm accessed on Sept 2010.
19. Tandon VR, Gupta RK, Vitex negundo Linn (VN) leaf extract as an adjuvant
therapy to standard anti-inflammatory drugs. Indian J Med Res. 2006; 124(4):
447-50.
20. Gupta RK, Tandon VR, Antinociceptive activity of Vitex-negundo Linn leaf
extract. Indian J Physiol Pharmacol. 2005; 49(2): 163-70.
21. Sathiamoorthy B, Gupta P, Kumar M, Chaturvedi A. K, Shukla PK, Maurya R.
New antifungal flavonoid glycoside from Vitex negundo. Bioorg Med Chem Lett.
2007; 17(1): 239-42.
22. Prabhu AR, Rajan AP, Review on the therapeutic potential of Vitex negundo Linn.
Journal of Pharmacy Research. 2010; 3(8): 1920-1922.
23. Devi PR, Kumari K, Kokilavani C, Effect of Vitex negundo leaf extract on the
free radicals scavengers in complete freund’s adjuvant induced arthritic rats.
Indian Journal of Clinical Biochemistry. 2007; 22 (1): 143-147.
24. Tiwari OP and Tripathi YB, Antioxidant properties of different fractions of Vitex
negundo Linn. Food Chemistry. 2007; 100(3): 1170-1176.
25. Tandon VR, Gupta RK. An experimental evaluation of anticonvulsant activity of
Vitex negundo. Indian J Physiol Pharmacol. 2005; 49(2): 199-205.
26. Alam MI, Gomes A, Snake venom neutralization by Indian medicinal plants
(Vitex negundo and Emblica officinalis) root extracts. J. Etanopharmacol. 2003;
86(1): 75-80.
27. Das S, Parveen S, Kundra CP, Pereira BM, Reproduction in male rats is
vulnerable to treatment with the flavonoid-rich seed extracts of Vitex negundo.
Phytother Res. 2004; 18(1): 8-13.
28. Sharma MM, Khanna P, Saini R, Batra A, Potential plant source of a promising
drug for cancer chemotherapy: Vitex negundo L. Journal of Economic and
Taxonomic Botany. 2006; 30: 269-273.
29. Tandon V, Gupta RK, Histomorphological changes induced by Vitex negundo in
albino rats. Indian Journal of Pharmacology. 2004; 36: 176-177.
30. Sheikh AT, Kaiser PJ, Gupta BD, Gupta VK, Johri RK, Negundoside, an irridiod
glycoside from leaves of Vitex negundo, protects human liver cells against,
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70
calcium-mediated toxicity induced by carbon tetrachloride. World J
Gastroenterol. 2008; 14(23): 3693-3709.
31. Villasenor IM, Lamadrid MR, Comparative anti-hyperglycemic potentials of
medicinal plants. J. Ethnopharmacol. 2006; 104(1-2):129-31.
32. Adnaik RS, Pai PT, Mule SN, Naikwade NS, Magdum CS. Laxative Activity of
Vitex negundo Linn. Leaves. Asian J. Exp. Sci. 2008; 22(1): 159-160.
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2.3 DIAGNOSTIC METHODS OF TUBERCULOSIS
Tuberculosis (TB) remains one of the major causes of death from a single
infectious agent worldwide of great concern for TB control is the emergence of drug
resistance. Since there is no cure for some multidrug-resistant strains of Mycobacterium
tuberculosis, there is concern that they may spread around the world, stressing the need
for additional control measures, such as new diagnostics, better drugs for treatment and
more effective vaccine.
Pulmonary TB can be diagnosed by its symptoms, chest radiography, sputum
smear microscopy and by cultivation of M. tuberculosis, which is considered as the gold
standard. Recent advances in molecular biology and molecular epidemiology, and a better
understanding of the molecular basis of drug resistance in TB have provided
new tools for
rapid diagnosis; however the high cost of most of these techniques and their requirement
for sophisticated equipment and skilled personnel have precluded their implementation
on
a routine basis, especially in low-income countries. Other nonconventional diagnostic
approaches proposed include the search for biochemical markers, detection of
immunological response and early detection of M. tuberculosis by methods other
than
colony counting. In this some of these methods reviewed
for their implementation in
diagnostic laboratories.
2.3.1 AUTOMATED CULTURE METHODS(2)
Although known for decades, liquid media for cultivation of mycobacteria had
never attracted the attention of mycobacteriologists. In fact, the ability of a liquid medium
to support a faster growth was heavily hampered by its susceptibility to contamination.
The use of antimicrobial combinations suitable of inhibiting the growth of the whole
spectrum of potential contaminants (Gram-positive and Gram-negative bacteria as well as
fungi) represented a turning point. During the same period, automation was taking its first
steps in microbiology, with blood cultures leading the field.
2.3.1.1 BACTEC TB-460(2,3,4,5.7,8,9,10,11,12,15,22)
The BACTEC TB-460 system (Becton Dickinson, Sparks, MD) was the first, and for
many years the only, automated approach in mycobacteriology. It makes use of a
radiometric instrumentation developed for blood cultures with the broth bottles replaced
by vials containing a medium specific for mycobacteria.
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The principle:
The medium: A modified Middlebrook 7H9 medium is used, in which one of the
components palmitic acid is radiolabeled with 14C. Contamination is controlled by the
addition, prior to use of a mixture of polymyxin B, amphotericin B, nalidixic acid,
trimethoprim and azlocillin (PANTA) reconstituted with a poly-oxyethylene solution. The
use of such a combination of antibiotics does not eliminate the decontamination step,
which needs to be performed before inoculation of the samples. The vials containing the
medium remain sealed through the whole culture process and the specimen is inoculated
by puncturing the rubber septum with a needle.
Instrumentation: Once the paired needles have perforated the rubber septum of the vial,
the gaseous phase is aspirated and replaced with air containing 5% CO2. The aspirated
gas is analyzed by a β-counter to quantify the eventual presence of radiolabeled CO2.
The rationale: When viable mycobacteria are present in the culture vial, the radiolabeled
palmitic acid is metabolized and radioactive CO2 is liberated into the gaseous phase.
The performance: The BACTEC TB-460 was first commercialized in 1980 and soon
became popular worldwide. It is not a fully automated system, as the vials, which are held
in an external incubator, must be loaded into the instrument for reading. The reading is
usually performed twice a week during the first 15 days of incubation, and weekly
thereafter until the 42nd day. BACTEC TB-460 is still used in many laboratories
worldwide.
Disadvantages:
1. The increasing cost of radioactive waste disposal.
2. The interest of the manufacturer to promote newly developed alternative systems.
2.3.1.2 The BACTEC MGIT 960 system(2, 6,7,8 ,13,14)
The BACTEC MGIT 960 system (Becton Dickinson, Sparks MD) uses the technology of
the previously developed blood culture instrument. The original system (BACTEC 9000)
was first adjusted to support mycobacterial cultures but was sequently completely
redesigned to process tubes.
The principle:
The medium: Mycobacteria Growth Indicator Tube (MGIT) is a modified Middlebrook
7H9 medium in which a supplement is added at the moment of use. The supplement is a
mixture of oleic acid, albumin, dextrose, and catalase (OADC) enrichment and the same
PANTA antibiotic mixture used in the radiometric system. The presence of PANTA does
not do away with the decontamination step, which needs to be done before inoculation.
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As the tubes containing the medium are screw-capped, no needle is needed for
inoculation. A silicon film embedded with a ruthenium salt is present at the bottom of the
tube as a fluorescence indicator
The instrumentation: Incubator and reader are combined in a single cabinet. The bottom
of each tube stimulated by ultraviolet light, is monitored by a fluorescence reader.
Fluorescence-emitting tubes are reported as positive. It is also possible to use the MGIT
tubes without instrumentation by holding the tubes in a normal incubator and observing
the fluorescence under a Wood’s lamp.
The rationale: The oxygen normally present in the medium quenches the natural
fluorescence of the ruthenium salt. If viable mycobacteria are present in the tube, oxygen
is consumed due to their metabolism, the quenching effect lowers accordingly and the
bottom of the tube fluoresces when exposed to ultraviolet light.
The performance: The BACTEC MGIT960 is a typical walk-away instrumentation
which monitors the tubes at one-hour intervals, alerts when they become positive and
signals the end of the incubation period.
Advantages: clearly faster and more sensitive than solid media.
2.3.1.3 VersaTREK/ESP system(2, 4,15,16)
The VersaTREK (previously known as the ESP system II) uses the technology of a
previously developed blood culture system and is commercialized by Trek Diagnostic
Systems.
The principle:
The medium: It uses a modified Middlebrook 7H9 medium to which the OADC
enrichment must be added. Two different antimicrobial mixtures are available. The first
one also known as AS includes polymyxin B, azlocillin, fosfomycin, nalidixic acid, and
amphotericin B. The second contains polymyxin B, vancomycin, nalidixic acid and
amphotericin B (PVNA). Usually, AS is used for specimens originating from sterile
samples or with a low risk of contamination, while PVNA is used for heavily-
contaminated samples. The presence of such antimicrobial mixtures for contamination
control does not eliminate the decontamination step which needs to be performed before
inoculating the sample. The bottles of medium hold a cellulose sponge whose large
surface area allready improves growth. Bottles are inoculated through a rubber septum by
means of a syringe.
The method: In the ESP susceptibility test system, lyophilized PZA powder (Trek
Diagnostics) was rehydrated with 25 ml of PZA reconstitution fluid according to the
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manufacturer's instructions and aliquots were frozen at -70°C. One milliliter of the
reconstituted drug was inoculated into an ESP Myco bottle.
The final concentration of
PZA in the test bottle was 300µg/ml. One milliliter of the rehydration fluid was
inoculated into the drug-free control bottle. The primary ESP Myco bottle served as the
inoculum source. Positive bottles may be used as the inoculum source up to 72 h after
producing a positive signal. An aliquot was removed from the ESP Myco bottle and
diluted 1:10 with normal sterile saline. A 0.5 ml aliquot of this suspension was used
to
inoculate the drug containing and control bottles. The control and drug-containing bottles
were placed into the ESP instrument, where they are monitored for changes in gas
pressure.
The instrumentation: Incubator and reader are combined in a single cabinet which also
shakes the bottles during the incubation. The pressure within each bottle is monitored by
a manometer through a proper connector. Cultures presenting a decreased headspace
pressure are reported as positive.
The rationale: If viable mycobacteria are present in the bottle, the oxygen consumption
due to their metabolism reduces the internal pressure. An isolate is considered resistant to
PZA if the drug-containing bottle signals positive within 3 days of the control bottle
becoming positive. Conversely, an isolate is reported to be susceptible to PZA if the drug-
containing bottle fails to signal within 3 days of the control becoming positive.
The performance: VersaTREK/ESP system is a typical walk-away instrumentation
which continuously monitors the bottles, alerts when they become positive and signals the
end of the incubation period.
Advantages:
1. VersaTREK in comparison with solid media and similar automated and semi-
automated competitor systems performs better than solid media but shows no
substantial advantage over other systems.
2. Mycobacterial blood cultures can also be performed with the VersaTREK system.
However, whole blood cannot be used and a previous treatment is required to
obtain sediment for inoculation.
2.3.1.4 BacT/Alert 3D ststem(2, 12,17,18 )
BacT/Alert 3D (previously known as MB/BacT) is commercialized by BioMérieux and
uses the technology of a previously developed blood culture system.
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The principle:
The medium: A modified Middlebrook 7H9 medium is used in which a supplement, a
mixture of OADC enrichment and polymyxin B, amphotericin B, nalidixic acid,
trimethoprim, vancomycin and azlocillin is added at the moment of use. The presence of
such contamination-controlling antibiotics does not eliminate the decontamination step
needed before inoculation. The bottles of medium have a CO2 sensor at the bottom and
are inoculated through a rubber septum by means of a syringe.
The instrumentation: Incubator and reader are combined in a single machine which does
not shake the bottles during incubation. The CO2 sensor is impacted by a light whose
reflected ray is monitored by a photodiode. Bottles producing specific changes in the
intensity of the reflected light are reported as positive.
The rationale: If viable mycobacteria are present in the bottle, the CO2 produced by their
metabolism causes a change in the color of the sensor, from green to yellow, which alters
the intensity of the reflected light ray.
Fig. no. 10 BacT/Alert technology
The performance: BacT/ALERT 3D is a typical walk-away instrumentation which
monitors the bottles at 10-min intervals, alerts when they become positive, and signals the
end of the incubation period.
Advantages:
1. It is faster and more sensitive than conventional media.
2. The system is also suitable for mycobacterial blood cultures, provided proper
bottles are used; no previous treatment of the blood is required.
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2.3.2 Nucleic acid amplification methods(2,19,22)
When the polymerase chain reaction (PCR) methodology took its first steps into
diagnostic microbiology, M. tuberculosis have the potential to benefit from this novel
technique. The rapid diagnosis of TB is possible by this method.
A] In house methods for diagnosis of tuberculosis(2,20)
One of the first findings on the way to developing a PCR technique aimed at M.
tuberculosis detection was that, although different targets were investigated, none of them
were suitable for differentiating M. tuberculosis from other species belonging to the M.
tuberculosis complex. In fact, the differentiation of such species is of very limited
relevance from the clinical and therapeutic point of view. Alternative amplification
methods were developed. Most successful were the reverse transcriptase PCR, the ligase
chain reaction and the strand displacement amplification.
B] Commercial methods(2,,22)
In the last few years, several amplification methods have been commercialized; only four
methods have gained worldwide diffusion and been widely validated by international
studies, although one of them (LCx, Abbott) is no longer on the market.
1. Amplified MTD(2,12,22)
Amplified Mycobacterium tuberculosis Direct Test (AMTD), developed by Gen-Probe
(San Diego, CA, USA), is an isothermal (42°C) transcriptase-mediated amplification
system.
2. Amplicor MTB Test (2,,21)
The Amplicor MTB Test (Roche Molecular Systems, Basel, Switzerland) relies on
standard PCR. The amplification and detection steps are carried out automatically by
the Cobas Amplicor instrument.
3. BD ProbeTec ET(2,23)
The BD ProbeTec ET (Becton Dickinson, Sparks, MD) uses DNA polymerase and
isothermal strand displacement amplification to produce multiple copies of IS6110, an
insertion element unique to M. tuberculosis complex.
Advantage: Specificity is good.
Disadvantages:
1. The unsatisfactory sensitivity is major limitation.
2. Paucibacillary specimens also detected by molecular amplification.
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3. Time consuming and also raises the risk of contamination due to which false
positive results.
4. The sediment of samples contains substances inhibiting the amplification process
and no method available for its neutralization.
2.3.3 Genetic identification methods (2)
Following the extraordinary development of molecular methods, the identification of
mycobacteria, previously based on phenotypic investigations, suddenly started to rely on
genotypic methods. Different genetic approaches developed in research laboratories
became rapidly popular in diagnostic laboratories and some of them were transformed
into commercial diagnostic kits.
1] PCR restriction-enzyme analysis(2,,24)
2] DNA probes: 2 A] AccuProbe (2,25)
2 B] Line probe assays: Three commercial methods are available,
A] INNO-LiPA MYCOBACTERIA (Innogenetics, Ghent, Belgium) (2, 11, 26, 27)
B] GenoType Mycobacterium (Hain, Germany)(28)
C] GenoType MTBC (Hain, Germany).
2.3.4 Non-conventional phenotypic diagnostic methods(2)
A] Phage-based assays:
The phage-based assay relies on the ability of M. tuberculosis to support the growth of an
infecting mycobacteriophage.
B] The micro-colony method(2,30,31)
The micro-colony method or thin-layer agar technique is an old method for culturing and
identifying mycobacteria; it allows both rapid detection and presumptive identification of
isolates based on the characteristic morphology of mycobacteria in cultures and has been
proposed as an inexpensive alternative method for the rapid detection and culture of
mycobacteria.
Advantages:
1. Sensitivity was 92.6% for thin layer 7H11 agar.
2. Time for detection of 11.5 days.
Disadvantages: The contamination rate was 5.1%.
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Fig. no. 11 Microcolonies of M. tuberculosis after, 4, 6, 8, and 15 days of culture
(Courtesy J. Robledo)
C] Microscopic observation broth-drug susceptibility assay (MODS) (2, 32 ,33,34)
The principle: The method is based on the observation of the characteristic cord
formation of M. tuberculosis visualized microscopically in liquid medium with the use of
an inverted microscope.
Method: Briefly, broth cultures were prepared in 24-well tissue-culture plates
(Becton
Dickinson), each containing 720 µl of decontaminant, Middlebrook 7H9 broth (Becton
Dickinson), oxalic acid, albumin, dextrose, and catalase (OADC) (Becton Dickinson) and
polymyxin, amphotericin B, nalidixic acid, trimethoprim and azlocillin
(PANTA) (Becton
Dickinson). For each sample, 12 wells were used: in 4 control wells, no drug was added,
and each of the remaining 8 wells contained one of four drugs at one of two
concentrations tested. The cultures were examined under an inverted light microscope
at a
magnification of 40x every day (except Saturday and Sunday) from day 4 to day 15, on
alternate days from day 16 to day 25, and twice weekly from day 26 to day 40.
The Rationale: Positive cultures were identified by cord formation, characteristic of M.
tuberculosis growth, in liquid medium in drug-free control wells.
Advantages:
1. The sensitivity for the detection of M. tuberculosis was 97.8 % compared to 89.0
% for the automated mycobacterial culture, and 84.0 % for Löwenstein-Jensen
medium (P < 0.001).
2. The median turnaround time was 7, 13 and 26 days for MODS, the automated
culture system and Löwenstein-Jensen medium, respectively (P < 0.001).
3. The method is rapid, inexpensive, sensitive and specific method for M.
tuberculosis detection and susceptibility testing.
Limitations: The requirement for an inverted microscope, which is necessary to observe
the cord formation in liquid medium.
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D] Analysis of cell wall mycolic acids(2,35)
Mycobacteria have unusually high lipid content in their cell wall. Such lipids include
mycolic acids and other saturated and unsaturated fatty acids. Mycolic acids are branched,
long-chain fatty acids present in the cell wall of a limited number of genera; they exhibit
the maximum length in the genus Mycobacterium. The analysis of the lipid content of the
mycobacterial cell wall has been widely used for identification purposes. The various
techniques used are based on the physical partitioning between two phases (stationary and
mobile) of single lipids present in the mycobacterial cell wall. The extraction of the lipids
from the bacterial colonies is the preliminary step in all the techniques described below.
1. Thin-layer chromatography (TLC)
2. Gas-Liquid Chromatography (GLC)
3. High-Performance Liquid Chromatography (HPLC)
Advantages:
1. This can be performed by thin-layer chromatography and by high-performance
liquid chromatography, that it can be completed in a few hours,
2. It is relatively inexpensive, and can identify a wide range of mycobacterial
species.
Disadvantage: The initial investment in the cost of the equipment.
Sensitivity Test Methods(36)
During the 1960s, there was much discussion about the methods used in drug
sensitivity tests (DSTs). The World Health Organization meetings reported three different
methods for performing DSTs; the absolute concentration method, the resistance ratio
method and the proportion method.
2.3.5 Proportion method(37,38,39)
Drug susceptibility of isolates to isoniazide, rifampicin, streptomycin and ethambutol was
performed by this method. Briefly, LJ media with drug incorporated in various
concentrations and plain LJ medium for control were prepared. The growth from a 3-4
week old culture was scraped with a loop and bacterial suspension was made in sterile
distilled water, vortexed and matched with McFarland opacity tube No. l. Dilutions of
10~2 and 10"3 were made and inoculated on both the control and drug containing media
and incubated at 370C. The first reading was taken after 28 days of incubation and the
second on 42 day. The percentage resistance (R) was calculated as the ratio of the number
of colonies on the drug containing media to those on the control medium.
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The Rationale:
R (%) = No. of colonies on drug media/ No. of colonies on control medium ×100
If R = >1 per cent, the isolate was taken as resistant.
A] Agar proportion method (37,38)
The agar proportion method, as recommended by the National Committee for Clinical
Laboratory Standards was performed to determine the percentage of M. tuberculosis
organisms resistant to each of the concentrations of antimycobacterial agents tested.
Briefly, 7H10 agar medium (Difco) was prepared, autoclaved at 121°C at 15 lb/in
2 for
15 min, and cooled to 50 to 56°C. INH, RIF, and EMB were added to the medium to yield
final concentrations of 0.2, 1.0, and 5.0 µg of INH/ml, 1.0 µg of RIF/ml, and 5.0 µg of
EMB/ml. Subsequently, 5.0 ml of each concentration of antimycobacterial containing
medium was dispensed into labeled quadrants of sterile petri dishes. One quadrant per
plate was reserved for 7H10 medium free of any antimycobacterial agent to serve as a
growth control. Upon solidification of the medium, the plates were inoculated with 0.1 ml
of 102 and 10
4 dilutions of a suspension of each M. tuberculosis isolate equivalent
to a
McFarland 1.0 standard. The inoculated plates were then incubated at 37°C in the
presence of 5% CO2 for 3 weeks.
The Rationale: Each strain was classified as susceptible to a drug if the number of
colonies that grew on the drug-containing medium was <1% of the number of colonies
grown on the control plate and resistant if the number of colonies was >10%. In cases
where two drug concentrations were tested in the agar proportion method, a strain was
classified as intermediate if it showed resistance to the lower drug concentration but
was
susceptible to the higher drug concentration.
2.3.6 The resistance ratio method (39)
The resistance ratio (RR) method utilizes the ratio of the minimum inhibitory
concentration (MIC) for the patients’ strain to the MIC of the drug-susceptible reference
strain, H37Rv, both tested in the same experiment. Inclusion of the reference strain in
each experiment is not only for quality control but also to standardize the results by
taking into account the test variations within certain permissible limits. This feature
makes the RR method the most expensive of the three conventional methods that use
solid media. Reading after 4 weeks of incubation defines “growth” on any slope as the
presence of 20 or more colonies, and MIC is defined as the lowest drug concentration in
the presence of which the number of colonies is less than 20. The range required for the
test strain is determined by the variation in the MIC of H37Rv, and by the need to
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determine a resistance ratio of 2 or less for sensitive strains and a resistance ratio of 8 or
more for resistant strains.
2.3.7 The absolute concentration method (39)
The absolute concentration method was used originally to determine the MIC of
isoniazide and of the drug that will inhibit growth, i.e. fewer than 20 colonies by the end
of 4 weeks by adding a carefully controlled inoculum of M. tuberculosis to the control
and drug-containing media. Media containing several sequential dilutions of each drug
are used and resistance is indicated by the lowest concentration.
2.3.8 Colorimetric methods(40)
1] Microdilution Resazurin Assay (MRA) (41,42,43,45)
Method: The REMA plate method was carried out as described by Palomino et al.
Briefly, the inoculum was prepared from a fresh colony on LJ in 7H9 medium
supplemented with 0.1% casitone, 0.5% glycerol, 10% OADC. A growth control
containing no antibiotic and a sterile control without inoculation were also prepared on
each plate. Two hundred microliters of sterile water was added to all perimeter wells to
avoid evaporation during incubation. Turbidity of inoculum adjusted to a turbidity
equivalent to that of a McFarland no. 1 standard and diluted 1:10. The microdilution test
was performed in 96-well plates. Two-fold dilutions of each drug were prepared in the
test wells in complete 7H9 broth, the final drug concentrations being: isoniazide 128-
0.00625 mg/L, rifampicin 128–0.0625 mg/L, streptomycin 128-0.125
mg/L and
ethambutol 128-0.25 mg/L. Twenty microlitres of each bacterial suspension was added to
180 µL of drug-containing culture medium. Control wells were prepared with culture
medium only and bacterial suspension only. The plates were sealed and
incubated for 7-14
days at 37°C. The range of concentrations tested for nicotinamide was 8-2000 mg/L. The
plate was covered, sealed in a plastic bag and incubated at 37°C in
normal atmosphere.
After 7 days of incubation, 30 µL of resazurin solution was added to each well and the
plate was reincubated overnight. A growth control
containing no antibiotic and a sterile
control without inoculation were also prepared on each plate. Two hundred microliters of
sterile water was added to all perimeter wells to avoid evaporation during incubation.
Then at day 8, a change in colour from blue (oxidized state) to pink (reduced state)
indicated the growth of bacteria.
The Rationale: MIC was defined as the lowest drug concentration that prevented
resazurin colour change from blue to pink. Each MIC was determined three times in
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duplicate experiments. Viable counting from control wells and from test wells was
performed onto 7H11 agar plates.
Advantages:
1. MRA is a rapid, inexpensive, low technology procedure, suitable for susceptibility
testing of first- and second-line anti-tubercular drugs and for screening new
antitubercular compounds against M. tuberculosis clinical strains.
2. The MRA method a safe, rapid and reliable assay; the resazurin solution we
employed in the microplate test. MRA did not need stabilizing agents, its stability
being always ensured by diluting the frozen stock solution when needed for
the
test.
3. By means of MRA the drug susceptibility of different Mycobacterium species,
including Mycobacterium avium, clinical strains can be evaluated.
2] MTT assay(43,44,45,55)
This method was carried out as described by Abate et al. A stock solution of MTT [3-
(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide] at a concentration of
5 g/L
was prepared in PBS, pH 6.8 and kept at 4°C in the dark. Formazan solubilization buffer
was prepared by mixing 1:1 (v/v) 20% sodium dodecyl sulphate (SDS) and a solution of
50% N, N-dimethylformamide (DMF). The inoculum was prepared as described above
for the REMA plate method and the drug concentration ranges used were the same.
Preparation of the 96-well plates was identical as described for the REMA plate. After 7
days of incubation at 37 °C, 10 µL of the MTT solution (5 g/L) was added to each well
and the plate was re-incubated overnight. If a violet precipitate (formazan) appeared, 50
µL of the SDS/DMF solution was added to the wells and the plate re-incubated for 3 h. A
change in colour from yellow to violet indicated growth of bacteria
and the MIC was
interpreted as in the REMA plate.
3] Microplate Alamar Blue assay(46,47,48,54)
Two hundred microliters of sterile deionized water was added to all outer-perimeter wells
of sterile 96-well plates to minimize evaporation of the medium in the test wells during
incubation. The wells received 100 µl of 7H9GC broth, one hundred microliters of
2×drug solutions should add. By using a multichannel pipette, 100 µl was transferred and
the contents of the wells were mixed well. Identical serial 1:2 dilutions were continued
100 µl of excess medium was discarded from the wells. Final drug concentration
ranges
were as follows: for INH, 0.031 to 8.0 µg/ml; for RMP, 0.0156-4 µg/ml in initial tests and
0.062-16 µg/ml on repeat testing; for SM, 0.125-32 µg/ml; and for EMB, 0.5-128 µg/ml.
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One hundred microliters of M. tuberculosis inoculum was added to the wells by using an
Eppendorf repeating pipette (yielding a final volume of 200 µl per well).
Thus, the wells
served as drug-free (inoculum-only) controls. The plates were sealed with Parafilm and
were incubated at 37°C for 5 days. Fifty microliters of a freshly prepared 1:1 mixture of
10×Alamar Blue (Accumed International, Westlake, Ohio) reagent and 10% Tween
80 was added to well. The plates were reincubated at 37°C for 24 h. If control well turned
pink, the reagent mixture was added to all wells in the microplate (if the well remained
blue, the reagent mixture would be added to another control well and the result would be
read on the following day). The microplates were resealed with Parafilm and were
incubated for an additional 24 h at 37°C, and the colors of all wells were recorded. A blue
color in the well was interpreted as no growth and a pink color was scored as growth. The
MIC was defined as the lowest drug concentration which prevented a color change from
blue to pink.
Advantages:
1. Rapid test and their use of low technology and their low cost.
2. Method of choice for drug susceptibility testing of M. tuberculosis.
2.3.9 Nitrate reductase assay (43,47,49,50,51,52,53,55)
NRA was performed as described by Golyshevskaia et al.11 and Angeby et aln. The
following critical concentrations were used: 0.2µg/ml for INH, 40 µg/ ml for RIF, 4
µg/ml for SM and 2.0 µg/ml for EMB.
Method: Fresh subculture (1 µl loops of bacteria) from isolates of M. tuberculosis grown
on LJ medium was taken and vortexed in 3ml of phosphate buffer saline (PBS, pH 7.4)
and turbidity was adjusted according to McFarland standard no.1. Part of the suspension
was diluted 1:10 in PBS. For each isolate, 0.2 ml of suspension was inoculated into the
tubes containing LJ medium with potassium nitrate (KNO3 and the antitubercular drugs;
0.2 ml of the 1:10 dilution was inoculated into drug free media (LJ media) containing
KNO3 which served as growth controls. Tubes in triplicate were incubated at 37°C for 14
days and 0.5 ml of a mixture of three reagents (25 µl of concentrated HCl, 50 µl of 2%
sulphanilamide and 50 µl of 1% n-1-napthyl-ethylenediamine dihydrochloride) was added
to one drug-free control tube after 7 days of incubation. If its colour changes to pink then
tubes with drugs were tested.
The Rationale: An isolate was considered resistant if there was colour change (pink or
deep red to violet) in the drug tube in question greater than in the 1:10 diluted growth
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control on the same day. If the tubes did not show any colour change and remains the
same, these were further incubated for 10 days and for 14 days.
Advantages:
1. It is rapid, inexpensive and easy to perform as it does not require much
instrumentation; it could be used routinely in laboratories in developing countries
for drug susceptibility testing of M. tuberculosis.
2. Apply to the test directly to microscopy positive sputa, thus drastically reducing
the time needed for detection.
2.3.10 Flow cytometric susceptibility testing(54,56,57)
Aliquots of 0.9 ml of each actively growing M. tuberculosis isolate were transferred to
2.0 ml polypropylene screw-cap microtubes. The tubes were inoculated with different
concentrations of standard drugs. Drug-free controls of M. tuberculosis were also
prepared by inoculating them with 0.1 ml sterile phosphate-buffered saline (pH 7.4).
Subsequently, the suspensions were incubated for 24, 48, or 72 h at 37°C in
an
environment of 5% CO2. After incubation, 0.2 ml of each assay suspension was removed
and placed in a sterile 2.0 ml screw-cap microtube containing 30 µl of 8%
paraformaldehyde (pH 7.4). Samples were then mixed and held at room temperature for
40 min before being analyzed with a Bryte HS flow cytometer with WinBryte software
(Bio-Rad Laboratories, Hercules, Calif.). After paraformaldehyde treatment,
M. tuberculosis cells were detected and differentiated from non-M. tuberculosis particles
in 7H9 medium by using forward and side angle light scatter signals. Electronic noise and
background particles in the 7H9 medium were excluded from analysis by adjusting the
threshold monitor listed on the WinBryte software program. Forward- and side-angle light
scattering was then used to analyze M. tuberculosis cells that were incubated with or
without antimycobacterial agents. For each sample acquired, the flow cytometer provided
a histogram profile relating to the number of M. tuberculosis organisms in each of
2,048 logarithmic channels of increasing light scattering, a measurement of the number of
M. tuberculosis events/microliter and a contour plot of forward- versus side-angle light
scattering. Five thousand events were acquired for each sample.
The rationale: An isolate was considered susceptible by the flow cytometric method if
the number of M. tuberculosis organisms (events) in the drug-containing medium was
reduced 25, 45, and 50% or more after exposure to EMB, INH, and RIF, respectively,
compared to the number of tubercle bacilli in the drug-free control.
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Advantages:
1. Increases biosafety and the assay does not depend on use of FDA for obtaining
results.
2. Instead, the numbers of mycobacteria in the assay suspensions with or without
exposure to antimycobacterial agents are determined 72 h after initiation
of testing.
3. This biologically safer procedure is still more rapid than the BACTEC-460
system.
4. Simplicity and low cost.
2.3.11 Agar Diffusion assay(58)
The common disc or well diffusion assays in antimicrobial assays of natural products are
used to evaluate extracts of new compounds, but are merely an indication that there is
growth inhibition at some unknown concentration along the concentration gradient.
Disadvantage: Mycobacteria having a very lipid rich, hydrophobic cell wall are often
more susceptible to less polar compounds.
2.3.12 Macro and micro agar dilution (58)
Testing of known concentrations of compounds in an agar medium allows for the
quantitation of activity and the determination of a MIC. Most mycobacteria including M.
tuberculosis will grow well Middlebrook 7H11 agar supplemented with oleic acid,
albumin, dextrose and catalase.
Disadvantage:-Required at least 18 days to visibly detect growth of the colonies.
2.3.13 Septi-check AFB method(59)
The Septi-check system consists of a capped bottle containing 30ml of middle-brook 7H9
broth under enhanced (5-8%) CO2. A paddle with agar media enclosed in a plastic tube
and enrichment broth containing glucose, glycerin, oleic acid, pyridoxal, catalase,
albumin, polyoxyethylene 40 stearate, azlocillin, nalidixic acid, trimethoprim, polymyxin
B, amphotericin B. One side of the paddle is covered with nonselective middlebrook
7H11agar, the reverse side is divided into two sections: one contains 7H11 agar with
para-nitro-a-acetylamino-b-hydroxypropiophenone for differentiation of M. tuberculosis
from other mycobacteria, the other section contains chocolate agar for detection of
contaminants.
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Advantages: Simultaneous detection of M. tuberculosis, non-tuberculous mycobacteria
(NTM), other respiratory pathogens and even contaminants and better result as compared
to other methods.
Summary:
With traditional methods, such as the indirect or direct proportion method, it could take 3
to 4 weeks to obtain susceptibility results The time needed to obtain these results
represents a potential danger to patients, health workers and the community. The
development of rapid and inexpensive new methods for resistance detection is an urgent
priority for the management of MDR-TB in the world, especially in low-resource
countries where most cases of MDR-TB occur. The turn around time (TAT) is important
in order for the patient to receive an appropriate treatment.
The BACTEC 460 system requires isotopes and heavy equipment, consequently is
not feasible in most resource-poor settings. The commercial MGIT system, the non
radiometric alternative method is reliable, but still expensive to implement in low-
resource countries. The three low cost methods: Nitrate reductase assay, MTT and
Resazurin have been successfully tested in previous studies and might become
inexpensive alternative methods for rapid and accurate detection of drug resistance of
MDR-TB strains. Owing to their higher level of agreement with the gold standard
methods, they seem to have the potential to provide rapid detection. These methods do
not need any sophisticated equipment, are simple to perform, reduce the time to achieve
results compared to the Proportion method and could be implemented in laboratories with
limited resources. One disadvantage of the MTT and resazurin assays is their biosafety,
since the plates use liquid medium and could generate aerosols. It has been shown
recently that this format can be adapted to screw-cap tubes to avoid this situation.
Conclusion
Using liquid medium-based culture systems such as the BACTEC 460 TB system
Mycobacteria Growth Indicator Tube MGIT 960, BacT/ALERT 3D or ESP Culture
System II to perform indirect susceptibility tests (require a pure culture of M.
tuberculosis), the results are available anywhere after 9 to 30 days. If susceptibility tests
are performed directly from clinical specimens with these systems, the time needed for
results is between 4 and 23 days. Although rapid, these methods require expensive
substrates and equipment and are therefore not feasible in most developing countries. The
results obtained with the MGIT system, nitrate reductase assay and the colourimetric
methods (MTT and Resazurin) were available on average in 10 days as with the
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BACTEC 460 TB system. The time of detection for each method was almost the same.
The INNO LIPA Rif TB kit was used to confirm the resistance pattern of these strains but
cannot be used in routine practice in low resource countries due to its high cost. A rapid
and inexpensive nitrate reductase assay (NRA) for the drug susceptibility testing (DST) of
M. tuberculosis.
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