Figure 3.1: Wrightia tinctoria (Roxb.) R. Br.(A) Flowering twig, (B) Fruit, (C) Flower

Figure 3.2: Photographs of Wrightia tinctoria (Roxb.) R. Br.Flowers

Figure 3.3:Photographs of Wrightia tinctoria (Roxb.) R. Br.Fruits

W. tinctoria seed fibers are unicellular yellowish-buff in color, 1.2-1.4 cm long, 25-27 mm in diameter having pointed apex and large lumen with thin wall. The fibers are easily distinguishable from cotton and jute as evident from physical properties. W. tinctoria seed fibers shows presence of lignified tissues and very less quantity of cellulose, and absence of true cellulose and cuticle layer. Oxidizing agents disintegrates the lignified walls. W. tinctoria seed fibers are inherently wettable, solublize easily in acidic and basic medium, tensile strength 1.25´10⁷ N/M² which may ensure easy dispersibility in wounds (Bigoniya and Rana, 2006).

W. tinctoria is commonly used as adulterant of an important antidysentric drug Holarrhena antidysentrica another apocynaceae plant (Chopra et al., 1956; Chopra et al., 1958). The therapeutic properties of W. tinctoria are similar to that of H. antidysentrica, which contains several steroidal alkaloids and is an established antidiarrhoeal drug (Glasby, 1975; Khan, 1987). W. tinctoria plants as well as leafs are smaller in size than H. antidysentrica. The Holarrhena has longer and greener leaves while the leaves of Wrightia are not as large or as green as former. W. tinctoria has white flavored flowers and dark brown to black colored root which are less bitter than H. antidysentrica. H. antidysentrica seeds have their air-filled hairs at the apex, while in Wrightia the tuft of air filled hairs is at the base of the seed. A comparative study was carried out on both the seeds by Jolly and Mechery, (1996) which included pharmacognostical and physiochemical evaluation. W. tinctoria barks can be differentiated from H. antidysentrica as the former is less bitter or taste less, soothing and color is reddish-brown (Atal and Sethi, 1962).

Traditional and Ethnopharmacological Uses

Sweet Indrajao is called dhudi (Hindi) because of its preservative nature. Supposedly a few drops of its sap in milk prevent curdling and enhance its shelf life, without the need to refrigerate. The wood of Sweet Indrajao is extensively used for all classes of turnery. It is made into cups, plates, combs, pen holders, pencils and bed stead legs. It is commonly used for making Chennapatna toys.

Fresh leaves are acrid, pungent and are chewed for relief from toothache (Kirtikar and Basu, 1975; The Wealth of India, 1976). Bark and seeds are astringent, acrid, thermogenic, carminative, digestive, stomachic, antidysenteric, constipating, depurative, anthelmintic, aphrodisiac, febrifuge and diuretic. They are useful in vitiated conditions of pitta and kapha, dyspepsia, bilious affections, flatulence, colic stomach pain, diarrhoea, leprosy, psoriasis, haemorrhoids, helminthiasis, fever, burning sensation and dropsy. The plant is very useful as stomachic, in the treatment of abdominal pain, skin diseases, as antidiarrhoeal and antihaemorrhagic (Nadkarni, 1976; Joshi et al., 1980; Singh et al., 1980; Shah and Gopal, 1988).

The leaves are applied as a poultice for mumps and herpes and sometimes they are also munched to relieve toothache. Tribes in Tamil Nadu applies “Vetpalai” a paste of the leaves, mixed with neem (nimba: Azadirachta indica A.Juss.) oil for eczema (Anesan et al., 2007).

In folk medicine, the dried and powdered roots of Wrightia along with Phyllanthus amarus (keezhanelli) and Vitex negundo (nochi) is mixed with milk and orally administered to women for improving fertility. The bark and seeds are effective against psoriasis and non-specific dermatitis. It has antiinflammatory and antidandruff properties and hence is used in hair oil preparations. Hill tribes use latex of the bark and unripe fruits for coagulating and solidifying milk. W. tinctoria produces thick and milky white latex. It is also said that Palakkad (Palghat), in Kerala got its name (pala = milky latex-bearing tree and kaadu = forest) due to the predominance of W. tinctoria trees in the drier forests (Ranjit Daniels et al., 2007).

All parts of the plant yield a purple dye called pala-indigo. Bark powder is very useful when taken every day in small quantity for indigestion, acidity and stomach pain. It is also used in chronic lung diseases and asthma. Massage of W. tinctoria powder reduces bleeding gum and fowl smell in gum pyorrhea. A decoction of the leaves and bark is rubbed over the body in dropsy. The bark is used as tonic and mineral supplement. The seeds possess aphrodisiac and anthelmintic properties (Pandey, 1992; The Wealth of India, 1976).

Traditional healers of Chhattisgarh use Indrajau both internally and externally in the treatment of about 16 diseases. Aqueous paste of Indrajau root is given to the patients having intestinal worm problem. The traditional healers mix Indrajau bark to Giloi decoction mixture to make it more effective. Germinated seeds of Idrajau are being recommended for patients suffering from jaundice. The bark is used externally in skin troubles. The healers prepare paste by mixing Indrajau bark powder with cow urine and apply it to effected parts. Indrajau bark is given with cow milk in urinary troubles and with dahi (curd) in renal calculi. In treatment of fever, Indrajau is a main ingredient in popular herbal combinations. The use of Indrajau in treatment of jaundice, fever and worm problem is very popular among the traditional healers (Oudhia, 2003).

Phytochemistry

The very first approach towards the separation of alkaloidal constituents of W. tinctoria by TLC was approached by P. D. Sethi (1970). Dried bark powder of W. tinctoria was extracted with ethyl alcohol-chloroform (1:4) mixture for separation of total alkaloids. Different solvent systems were tried to separate individual alkaloids by TLC using Modified Dragendorff’s spraying reagent. Maximum six spots have been revealed and best separation has been effected with benzene-chloroform-ethyl alcohol (4:2:1) on alkaline silica gel G layer.

Reddyet al. (1999) carried out pharmacognostical studies on W. tinctoria bark to establish standards for identification of the drug. Macroscopy, histology, microchemical and phytochemical tests along with ash and extractive values were reported. Successive cold extraction of dried W. tinctoria bark collected from Mothapalayam of Nilgiri hills, Tamil Nadu, India in the month of June showed presence of the following constituents in qualitative phytochemical tests – Petroleum ether extract: steroids; chloroform extract: steroids and triterpenoids; ethylacetate extract: steroids, triterpenoids, saponins, flavonoids, tannins and phenolics; acetone and methanol extract: glycosides, steroids, triterpenoids, saponins, carbohydrates and flavonoids, tannins and phenolics. All extracts gave negative tests for alkaloids, fixed oils, cumarins, gums and resins.

The stem bark of W. tinctoria contains β-amyrin, lupeol, β-sitosterol and a new triterpenoid as reported by Rangaswami and Rao, (1963). The powdered bark was exhausted by cold percolation with petroleum ether. The petroleum ether extract contained a lot of rubbery material elimination of which gives one triterpene compound obtained by direct fractional crystallization from ethyl alcohol, but that cannot be identified (m.p. 170-72°C). The mother liquors after suitable fractionation technique yield β-amyrin acetate (m.p. 238-40°C), lupeol benzoate (m.p. 266-70°C) and β-sitosterol (m.p. 132-34°C). The identity has been established by color reactions, elementary analysis, optical rotation and mixed melting point with authentic sample.

Triacontanol and tryptanthrin (m.p. 265-67°C) have been isolated from W. tinctoria leaves as reported by George et al. (1996). β-amyrin benzoate (m.p. 229-31°C) was isolated from the petroleum ether extract of powdered leaves.

The powdered pods (freed from seeds), were extracted with hexane followed by hot chloroform. The hexane extract gave a mixture of terpenoids. Ursolic acid (m.p. 280-82°C) was obtained from chloroform extract of pods besides another triterpene acid (m.p. 130-31°C) from mother liquors which cannot be identified (Rao et al., 1966). In continution to their studies Rao et al. (1968) reported isolation of α-amyrin (m.p. 182-84°C), β-sitosterol and ursolic acid from hexane extract of pods. oleanolic acid (m.p. 250-53°C) was reported to isolate from mother liquors of ursolic acid.

Figure 3.4:Isolated Compound of Wrightia tinctoria

Column chromatography (petroleum ether, benzene and chloroform) of methanol extract of W. tinctoria immature dried seed pods gave five compounds. Four of these compounds were identified as cycloartenone, b-amyrin, cycloeucalenol and b-sitosterol and fifth one was identified as wrightial (m.p. 99°C), a new terpene. Petroleum ether–chloroform elute (1:1) gave a colourless compound identified as cycloartenone (m.p. 102-103°C) and a white solid which was identified as b-amyrin (m.p. 196°C). The chloroform elute gave colourless solid, wrightial (m.p. 99°C), cycloeucalenol (m.p. 140°C) and β-sitosterol (Ramchandra et al., 1993).

Isolation and characterization of a new sterol 14α-methylzymosterol in addition to four rare plant sterols, desmosterol (m.p. 95-97°C), clerosterol (m.p. 122-24°C), 24-methylene-25-methylcholesterol (m.p. 145-48°C) and 24-dehydropollinastanol (m.p. 75-78°C) have been reported from W. tinctoria seeds. Air dried and powdered seeds were extracted with petrol in a soxhlet extractor. Preparative TLC of the unsaponifiable lipid (petrol extract) gave sterol fraction which was further separated by argentation TLC, final isolation of each component from individual fractions was performed by HPLC (Akihisa et al., 1988).

W. tinctoria seeds contain 30 per cent of oil out of which 70 per cent is hydroxy acid. Oil is rich in isoricinoleic acid or 9-hydroxy-cis-12-octadecenoic acid (strophanthus acid) like some other plants of Apocynaceae family (Everett et al., 1981). Ahmad and Lie Ken Jie, (2008) carried out derivatization of methyl 9-hydroxyoctadec-12-ynoate to obtain the industrially important fatty compounds. Methyl 9-hydroxyoctadec-12-ynoate was subjected to mesylation and demesylation reactions in which an unknown compound methyl 9-ethoxyoctadec-12-ynoate was furnished in good yield, in addition to an inseparable mixture of enynoic esters. In another set of reactions methyl 9-hydroxyoctadec-12-ynoate was oxidized with trimethyl chlorochromate wherein the resulting methyl 9-oxooctadec-12-ynoate was subjected to three condensation reactions–with 1,2-ethanedithiol, ethylene glycol and b-mercaptoethanol separately–to yield diethyl 9,9-ethylenedisulfide octadec-12-ynoate, 9,9-ethylenedioxyoctadec-12-ynoate and 9-oxathiolaneoctadec-12-ynoate respectively in excellent yields. Their structures have been established using infrared (IR) spectroscopy, ¹H nuclear magnetic resonance (NMR) spectroscopy, ¹³C NMR spectroscopy and mass spectral studies.

Rajeshet al. (2007) compared the clot inducing and dissolving properties of Calotropis gigantea R. Br. (Asclepiadaceae), Synadenium grantii Hook. f. (Euphorbiaceae) and Wrightia tinctoria R. Br. (Apocynaceae) latex extracts. All the three latex extracts hydrolyzed casein, fibrinogen and crude fibrin dose-dependently. The proteolytic action on fibrinogen subunit was in the order of alpha > beta > gamma. All extracts exhibited procoagulant activity as assayed by re-calcification time. However thrombin like activity is restricted to C. gigantea. In addition the extracts dose-dependently hydrolyzed blood and plasma clots. Furthermore, the hydrolyzing pattern of fibrin in the plasma clot was substantiated by SDS-PAGE. The extracts hydrolyzed all the subunits (alpha polymer, alpha-chains, gamma-gamma dimer and beta-chain) of fibrin efficiently. Both fibrinogenolytic and fibrinolytic activity potency of the extracts were in the order of C. gigantea > S. grantii > W. tinctoria. S. grantii and W. tinctoria latex extracts were non-toxic and did not induce any hemorrhagic effect at the tested dose (> 200 microg). The proteolytic activities of S. grantii and W. tinctoria were inhibited by PMSF. Thus, this study provides the basis for the probable action of plant latex proteases to stop bleeding and effect wound healing as exploited in folk medicine.

A new protease named “wrightin” was purified from the latex of the plant W. tinctoria by cation-exchange chromatography. The enzyme is a monomer having a molecular mass of 57.9 kDa (MALDI-TOF), an isoelectric point of 6.0 and an extinction coefficient (1 per cent; 280) of 36. 4. Optimun activity is achieved at a pH of 7.5-10 and a temperature of 70°C. Wrightin hydrolyzes denatured natural substrates such as casein, azoalbumin, and hemoglobin with high specific activity; for example, the Km value is 50 mM for cadein as substrate. Wrightin showed weak amidolytic activity toward l-Ala-Ala–p-nitroanilide but completely failed to hydrolyze N-alpha-benzoyl–dl-arginine–p-nitroanilide (BAPNA), a preferred substrate for trypsin-like enzymes. Complete inhibition of enzyme activity by serine protease inhibitors such as PMSF and DFP indicates that the enzyme belongs to the serine protease class. The enzyme was not inhibited by SBTI and resists autodigestion. Wrightin is remarkably thermostable, retaining complete activity at 70°C after 60 min of incubation and 74 per cent of activity after 30 min of incubation at 80°. Besides, the enzyme is very stable over a broad range of pH from 5.0 to 11.5 and remains active in the presence of various denaturants, surfactants, organic solvents and metal ions. Thus wrightin might be a potential candidate for various applications in the food and biotechnological industries, especially in operations requiring high temperatures (Tomar et al., 2008).

Pharmacology

W. tinctoria bark is effective in psoriasis and nonspecific dermatitis. The leaves are used in various skin disorders including herpes. Oil emulsion of W. tinctoria pods is used to treat psoriasis (Anonymous, 1987). Psoriasis is characterized by epidermal hyperplasia and a greatly accelerated rate of epidermal turnover. The lesions are characteristically dry, well demarcated, red, slightly raised and scaly. Gentle scraping of the lesion removes scales and produces many pinpoint bleeding sites, the so-called “Auspitz sign”. The lesions are discrete or confluent erythromatous plaques and papules covered with white or silvery scales found on the extensor surfaces such as the elbows, knees, back and scalp.

Krishnamurthyet al. (1981) carried out clinical study of vetpalai (Wrightia tinctoria L.) oil in the treatment of kalanjagapadai (psoriasis). Alam et al. (1985) reported effect of insulation on “777 oil” used for psoriasis in Siddha system of medicine. According to Siddha system the fat soluble material of the leaves of W. tinctoria (Roxb.) R.Br. was extracted into the oil in sunlight, this method is called suryaputa. The fat soluble substances are claimed to have keratolytic action. Phytochemical studies carried out on the extract exposed to sunlight for four hours showed an increase in acid and iodine values. Process and product standardisation of “777 oil” used for psoriasis in Siddha medicine was reported by Alam et al. (1986). 777 oil was prepared from the leaves of W. tinctoria (Roxb.) R.Br. by insolation with coconut or gingelly oil as a base. In this study the drug prepared by insolation (exposure to sunlight) was compared with that prepared in darkness. The insolated drug had a higher iodine number, indicating the production of more unsaturated compounds.

A US patent has been issued by the title “Herbal medication for the treatment of psoriasis” on January 12, 1999. The invention includes a composition comprising a latex extracted from the leaves of the W. tinctoria R. Br. plant, water, urea, and polyethylene glycol for topical treatment of skin disorders, particularly psoriasis. The pharmaceutical preparation is a hydrophilic ointment that is capable of delivering the active drugs without being greasy or irritating to the skin (Herbal medication for the treatment of psoriasis, US). The antibacterial screening of the various extracts of the seeds of H. antidyssenterica and W. tinctoria were carried out and the chloroform and methanolic extracts of both the seeds were found to possess antibacterial activity (Jolly and Mechery, 1996).

Muruganandamet al. (1998a) reported effect of W. tinctoria leaf methanolic extract on anxiety patterns in rats. The effect of acute administration of W. tinctoria leaves methanolic extractives, constituting indigotin (HPTLC, relative abundance 21.97 per cent), indirubin (27.13 per cent), tryptanthrin (21 per cent), isatin (2.70 per cent) and rutin (14.24 per cent), was studied on the rat brain concentrations of monoamines and their metabolites in five different brain regions, viz. hypothalamus, hippocampus, striatum, pons medulla and frontal cortex. W. tinctoria extract was administered at the doses of 25 and 50 mg/kg, i.p. and the brain monoamines were assayed after 30 minutes of the treatment. W. tinctoria treatment significantly and dose dependently decreased the levels of serotonin (5-HT), its metabolite 5-hydroxy indole acetic acid (5-HIAA) and their turnover in all the brain regions assayed. On the other hand, extract treatment significantly and dose dependently augmented the levels of norepinephrine (NE), its metabolite methyl hydroxy phenyl glycol (MHPG) and also the turnover in all the brain regions studied. Similarly, the levels of dopamine (DA) was also significantly augmented in the hypothalamus, striatum and frontal cortex. Likewise, the levels of dihydroxy phenyl acetic acid (DOPAC), a metabolite of DA, was also increased in hypothalamus and frontal cortex. However, the treatment produced a significant decrease in the DOPAC in striatum. This differential modulation of the neurotransmitters and their metabolites can explain the behavioral effects of W. tinctoria, namely anxiolytic and antidepressant effect (Muruganandam et al., 1998b).

An experimental histological evaluation of W. tinctoria was conducted on reversal of parakeratosis to orthokeratosis based on mouse tail test. W. tinctoria

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