2.1 PLANT PROFILE OF RICINUS COMMUNIS - Shodhgangashodhganga.inflibnet.ac.in/bitstream/10603/23543/8/08_chapter_02.pdf · Jupiter and Leda. According to E. A. Weiss, writing in castor,

CHAPTER -2 LITERATURE REVIEW

Phytochemical and pharmacological investigation of anti-tubercular activity of the certain indigenous plants

36

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



37

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.



38

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



39

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)

.



40

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



41

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.



42

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.



43

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



44

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



45

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)

.



46

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.



47

• 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



48

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)

.



49

REFERENCES:

1. http://ntbg.org/plants/plant_details.php?rid=812&plantid=11833 accessed on July

2006.

2. http://en.wikipedia.org/wiki/Castor_bean castor oil plant accessed on July 2008.

3. The Ayurvedic pharmacopeia of India, Government of India Ministry of Health

and Family Welfare Department of Ism & H. 2001; Part-I Vol. 3: 49-50.

4. http://www.botanical.com/botanical/mgmh/c/casoil32.html accessed on Aug 2009.

5. http://waynesword.palomar.edu/plmar 99.htm accessed on Aug 2009.

6. http://www.inchem.org/documents/pims/plant/ricinus.htm accessed on June 2009.

7. http://server9.webmania.com/users/pfafardea/database/plants.php?Ricinus+comm

unis. accessed on Oct 2010.

8. Wealth of India, A dictionary of Indian raw materials and Industrial products. Ist

supplement series Vol.5, R-2 NISCAIR-2004; CSFR: 12-18.

9. Duke JA, Review on castor: Ricinus communis Linn. Euphorbiaceae Castorbean

Handbook of Energy Crops. 1983.

10. Kirtikar and Basu, Indian medicinal plants. IInd edition. Vol.III. 1991; 2272-2275.

11. http://www.ikisan.com/links/apcastorMorphology%20And%20Growth.shtml

accessed on June 2009.

12. http://www.hort.purdue.edu/newcrop/dukeenergy/ricinuscommunis.html accessed

on July 2008.

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.



50

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.



51

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,



52

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



53

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



54

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).



55

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,



56

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).



57

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



58

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.



59

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.



60

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.



61

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



62

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)

.



63

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



64

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



65

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)

.



66

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)



67

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).



68

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



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,



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.



71

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.



72

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.



73

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



74

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.



75

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.



76

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.



77

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%.



78

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.



79

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.



80

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



81

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



82

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.



83

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



84

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.



85

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.



86

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



87

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.



88

REFERENCES:

1. Global tuberculosis control. WHO Report 2009, 32.

2. Enrico Tortoli and Juan Carlos Palomino. New Diagnostic Methods.

www.tuberculosis textbook.com accessed on June 2008.

3. Miller MA, Thibert L, Desjardins F, Siddiqi SH, Dasca A, Testing of susceptibility of

Mycobacterium tuberculosis to pyrazinamide: comparison of Bactec method with

pyrazinamidase assay. Journal of Clinical Microbiology. 1995; 33(9): 2468-2470.

4. Vincent J. LaBombardi. Comparison of the ESP and BACTEC Systems for Testing

Susceptibilities of Mycobacterium tuberculosis Complex Isolates to Pyrazinamide.

Journal of Clinical Microbiology. 2002; 40(6): 2238-2239.

5. Roberts GD, Goodman NL, Heifets L, et al. Evaluation of the BACTEC radiometric

method for recovery of mycobacteria and drug susceptibility testing of

Mycobacterium tuberculosis from acid-fast smear-positive specimens. Journal of

Clinical Microbiology. 1983; 18: 689-696.

6. Hanna BA, Walters SB, Bonk SJ, Tick LJ. Recovery of mycobacteria from blood in

mycobacteria growth indicator tube and Löwenstein-Jensen slant after lysis-

centrifugation. Journal of Clinical Microbiology. 1995; 33 (3): 315–316.

7. Badak FZ, Kiska DL, Setterquist S, Hartley C, Connell MA, Hopfer RL. Comparison

of mycobacteria growth indicator tube with BACTEC 460 for detection and recovery

of mycobacteria from clinical specimens. Journal of Clinical Microbiology. 1996;

34: 2236-2239.

8. Pfyffer GE, Welscher HM, Kissling P, et al. Comparison of the Mycobacteria Growth

Indicator Tube (MGIT) with radiometric and solid culture for recovery of acid-fast

bacilli. Journal of Clinical Microbiology. 1997; 35: 364-368.

9. Siddiqi SH, Libonati JP, Middlebrook G. Evaluation of rapid radiometric method for

drug susceptibility testing of Mycobacterium tuberculosis. Journal of Clinical

Microbiology. 1981; 13: 908–912.

10. Evans KD, Nakasone AS, Sutherland PA, Maza LM, Peterson EM. Identification of

Mycobacterium tuberculosis and Mycobacterium avium- M. intracellulare directly

from primary BACTEC cultures by using acridinium-ester labelled DNA probes.

Journal of Clinical Microbiology. 1992; 30: 2427-2431.

11. Miller N, Infante S, Cleary T, Evaluation of the LiPA MYCOBACTERIA assay for

identification of mycobacterial species from BACTEC 12B bottles. Journal of




89

12. Tortoli E, Mattei R, Savarino A, Bartolini L, Beer J, Comparison of Mycobacterium

tuberculosis susceptibility testing performed with BACTEC 460TB (Becton

Dickinson) and MB/BacT (Organon Teknika) systems. Diagn Microbiol Infect Dis.

2000; 38: 83–86.

13. Alaci, M, Ueki SY, Sato DN, Da Silva Telles MA, Curcio M, Silva EA. Evaluation of

mycobacteria growth indicator tube for recovery and drug susceptibility testing of

Mycobacterium tuberculosis isolates from respiratory specimens. Journal of Clinical

Microbiology. 1996; 34:762-764.

14. Palomino JC, Traore H, Fissette K, Portaels F, Evaluation of Mycobacteria Growth

Indicator Tube (MGIT) for drug susceptibility testing of Mycobacterium tuberculosis.

Int. J. Tuberc. Lung Dis. 1996; 3: 344-348.

15. Ruiz P, Zerolo FJ, Casal MJ, Comparison of susceptibility testing of Mycobacterium

tuberculosis using the ESP culture system II with that using the BACTEC method.

Journal of Clinical Microbiology. 2000; 38:4663-4664.

16. Woods GL, Fish G, Plaunt M, Murphy T, Clinical evaluation of difco ESP culture

system II for growth and detection of mycobacteria. Journal of Clinical

Microbiology. 1997; 35: 121-24.

17. Diaz-Infantes MS, Ruiz-Serrano MJ, Martinez-Sanchez L, Ortega A, Bouza E.

Evaluation of the MB/BacT mycobacterium detection system for susceptibility testing

of Mycobacterium tuberculosis. Journal of Clinical Microbiology. 2000; 38: 1988-

1989.

18. Brunello F, Fontana R. Reliability of the MB/BacT system for testing susceptibility of

Mycobacterium tuberculosis complex isolates to anti-tuberculous drugs. Journal of


19. Simboli N, Takiff H, McNerney R, et al. In-house phage amplification assay is a

sound alternative for detecting rifampin-resistant Mycobacterium tuberculosis in low-

resource settings. Antimicrob Agents Chemother 2005; 49: 425-427.

20. Noordhoek GT, van Embden JD, Kolk, AH. Reliability of nucleic acid amplification

for detection of Mycobacterium tuberculosis: an international collaborative quality

control study among 30 laboratories. Journal of Clinical Microbiology. 1996; 34:

2522-2525.

21. Alovisio JR, Montenegro-James S, Kemmerly SA, Comparison of the Amplified

Mycobacterium tuberculosis (MTB) direct test, Amplicor MTB PCR, and IS6110-



90

PCR for detection of MTB in respiratory specimens. Clin Infect Dis 1996; 23: 1099-

1106.

22. CDC. Nucleic acid amplification tests for tuberculosis. MMWR. 2000; 49: 593-594.

23. Bergmann JS, Keating WE, Woods GL, Clinical evaluation of the BDProbeTec ET

system for rapid detection of Mycobacterium tuberculosis. Journal of Clinical

Microbiology. 2000; 38: 863-865.

24. Ruiz M, Torres MJ, Llanos AC, Arroyo A, Palomares JC, Aznar J. Direct detection of

rifampin- and isoniazid-resistant Mycobacterium tuberculosis in auramine-rhodamine-

positive sputum specimens by real-time PCR. Journal of Clinical Microbiology.

2004; 42: 1585-1589.

25. Sougakoff W, Rodriguez M, Truffot-Pernot C, et al. Use of a high-density DNA

probe array for detecting mutations involved in rifampicin resistance in

Mycobacterium tuberculosis. Clin Microbiol Infect. 2004; 10: 289-294.

26. Suffys PN, da Silva Rocha A, de Oliveira M, et al. Rapid identification of

Mycobacteria to the species level using INNO-LiPA Mycobacteria, a reverse

hybridization assay. Journal of Clinical Microbiology. 2001; 39: 4477-4482.

27. Rossau R, Traore H, De Beenhouwer H, et al. Evaluation of the INNO-LiPA Rif. TB

assay, a reverse hybridization assay for the simultaneous detection of Mycobacterium

tuberculosis complex and its resistance to rifampin. Antimicrob Agents Chemother.

1997; 41: 2093-2098.

28. Ruiz P, Gutierrez J, Zerolo FJ, Casal M, GenoType mycobacterium assay for

identification of mycobacterial species isolated from human clinical samples by using

liquid medium. Journal of Clinical Microbiology. 2002; 40: 3076-3078.

29. Mejia GI, Castrillon L, Trujillo H, Robledo JA. Microcolony detection in 7H11 thin

layer culture is an alternative for rapid diagnosis of Mycobacterium tuberculosis

infection. Int J Tuberc Lung Dis. 1999; 3: 138–142.

30. Caviedes L, Lee TS, Gilman RH, et al. Rapid, efficient detection and drug

susceptibility testing of Mycobacterium tuberculosis in sputum by microscopic

observation of broth cultures. Journal of Clinical Microbiology. 2000; 38: 1203-

1208.

31. Park WG, Bishai WR, Chaisson RE, Dorman SE. Performance of the microscopic

observation drug susceptibility assay in drug susceptibility testing for Mycobacterium

tuberculosis. Journal of Clinical Microbiology. 2002; 40: 4750-4752.



91

32. David AJ, Moore MD, Carlton AW, Microscopic-Observation Drug- Susceptibility

Assay for the Diagnosis of TB, Journal of Clinical Microbiology. 2006; 355(15):

1539-1550.

33. Moore DA, Mendoza D, Gilman RH, et al. Microscopic observation drug

susceptibility assay, a rapid, reliable diagnostic test for multidrug-resistant

tuberculosis suitable for use in resource-poor settings. Journal of Clinical

Microbiology. 2004; 42: 4432-4437.

34. Butler WR, Guthertz LS. Mycolic acid analysis by high-performance liquid

chromatography for identification of Mycobacterium species. Clin Microbiol Rev.

2001; 14: 704-726.

35. Albert H, Trollip A, Seaman T, Mole RJ, Simple, phage-based (FASTPplaque)

technology to determine rifampicin resistance of Mycobacterium tuberculosis directly

from sputum. Int J Tuberc Lung Dis. 2004; 8: 1114-1119.

36. Mitchison DA, Drug resistance in tuberculosis. European Respiratory Journal. 2005;

377-379.

37. Canetti G, Fox W, Khomenko AL et al. Advances in techniques of testing

mycobacterial drug sensitivity and the use of sensitivity tests in tuberculosis control

programs. Bull World Health Organ. 1969; 41, 21-43.

38. Aziz MA, Mario AL, Raviglione guidelines for surveillance of drug resistance in

tuberculosis 2nd Edition, WHO, Geneva WHO/TB/2003; 320: 30-31.

39. Yajko DM, Madej JJ, Lancaster MV, et al. Colorimetric method for determining

MICs of antimicrobial agents for Mycobacterium tuberculosis. Journal of Clinical

Microbiology. 1995; 33: 2324-2327.

40. Palomino JC, Martin A, Camacho M, Guerra H, Swings J, Portaels F. Resazurin

microtiter assay plate: simple and inexpensive method for detection of drug resistance

in Mycobacterium tuberculosis. Antimicrob Agents Chemother. 2002; 46: 2720-

2722.

41. Elena BE, Giuditta SG, Bragadin CM, Development of a microdilution method to

evaluate Mycobacterium tuberculosis drug susceptibility, Journal of Antimicrobial

Chemotherapy. 2003; 52, 796-800.

42. Martin A, Camacho M, Portaels F, Palomino JC, Resazurin microtiter assay plate

testing of Mycobacterium tuberculosis susceptibilities to second-line drugs: rapid,

simple, and inexpensive method. Antimicrob Agents Chemother. 2003; 47: 3616-

3619.



92

43. Montoro E, Lemus D, Echemendia M, Martin A, Portaels F, Carlos J, Palomino

Comparative evaluation of the nitrate reduction assay, the MTT test and the resazurin

microtitre assay for drug susceptibility testing of clinical isolates of Mycobacterium

tuberculosis. Journal of Antimicrobial Chemotherapy. 2005; 55(4): 500-505.

44. Abate G, Mshana RN, Miorner H, Evaluation of a colorimetric assay based on 3-(4, 5-

dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide (MTT) for rapid detection of

rifampicin resistance in Mycobacterium tuberculosis. Int J Tuberc Lung Dis. 1998;

2: 1011-1016.

45. Martin A, Takiff H, Vandamme P, Swings J, Carlos J, Palomino and Françoise

Portaels, A new rapid and simple colorimetric method to detect pyrazinamide

resistance in Mycobacterium tuberculosis using nicotinamide, Journal of

Antimicrobial Chemotherapy. 2006; 58(2): 327-331.

46. Scott GF, Witzig RS, McLaughlin JC, et al, Rapid, Low-Technology MIC

Determination with Clinical Mycobacterium tuberculosis Isolates by Using the

Microplate Alamar Blue Assay, Journal of Clinical Microbiology. 1998; 36(2): 362-

366.

47. Kumar M, Khan IA, Verma V, Qazi GN, Nitrate reductase assay versus Microplate

Alamar Blue assay for MIC determination of Mycobacterium tuberculosis. Int J

Tuberc Lung Dis, 2005; 9: 939-941.

48. Sungkanuparph S, Pracharktam R, Thakkinstian A, Buabut B, Kiatatchasai W.

Correlation between susceptibility of Mycobacterium tuberculosis by microtiter plate

Alamar Blue assay and clinical outcomes. J Med Assoc Thai. 2002; 85: 820-824.

49. Sethi S, Sharma S, Sharma SK, Meharwal SK et al, Drug susceptibility of

Mycobacterium tuberculosis to primary antitubercular drugs by nitrate reductase

assay. Indian Journal of Medical Research. Nov 2004.

50. Lemus D, Montoro E, Echemendía M, Martin A, Portaels F, Palomino JC, Nitrate

reductase assay for detection of drug resistance in Mycobacterium tuberculosis:

simple and inexpensive method for low-resource laboratories, Journal of Clinical

Microbiology. 55; 55: 861-863.

51. Humberto RM, Marta A, Alejandro S, Kristian Angeby KA, Drug susceptibility

testing of Mycobacterium tuberculosis by a nitrate reductase assay applied directly on

microscopy-positive sputum samples, Journal of Clinical Microbiology. 2005; 43(7):

3159-3161.



93

52. Angeby KA, Klintz L, Hoffner SE, Rapid and inexpensive drug susceptibility testing

of Mycobacterium tuberculosis with a nitrate reductase assay, Journal of Clinical

Microbiology. 2004; 553-555.

53. Coban AY, Birinci A. Ekinci B. Durupinar B, Drug susceptibility testing of

Mycobacterium tuberculosis with nitrate reductase assay. Int J Antimicrob Agents.

2004; 24: 304-306.

54. Reis RS, Neves IJ, Lourenco SL, Fonseca LS, Lourenco MC, Comparison of flow

cytometric and Alamar Blue tests with the proportional method for testing

susceptibility of Mycobacterium tuberculosis to rifampin and isoniazide. Journal of


55. Heifets LB, Iseman MD, Cook JL, Lindholm-Levy PJ, and Drupa I. Determination of

in vitro susceptibility of Mycobacterium tuberculosis to cephalosporins by

radiometric and conventional methods. Antimicrob. Agents Chemother. 1985; 27:

11-15.

56. Kirk SM., Schell RF, Moore AV, Callister SM, Mazurek GH, Flow cytometric testing

of susceptibilities of Mycobacterium tuberculosis isolates to ethambutol, isoniazide,

and rifampin in 24 hours. Journal of Clinical Microbiology. 1998; 36: 1568-1573.

57. Norden MA, Kurzynski TA, Bounds SE, Callister SM, Schell RF. Rapid susceptibility

testing of Mycobacterium tuberculosis (H37Ra) by flow cytometry. Journal of

Clinical Microbiology. 1995; 33: 1231-1237

58. Gautam R, Saklani A, Jachak SM. Indian medicinal plants as a source of

antimycobacterial agents. Journal of Ethanopharmacology. 2007; 110-202.

59. What is new in the diagnosis of Tuberculosis? ICMR bulletin 2002; 32(8): 61.

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