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Phytoconstituents and HPTLC analysis in Saraca asoca (Roxb.)Wilde

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Saraca asoca (Roxb.)Wilde, (Ashok) belonging to the family Caesalpiniaceae is a universal panacea in the ayurvedic medicine. This versatile plant has anti-cancer, anti-menorrhagic, anti-oxytocic, anti–microbial activity and has extended uses in ayurveda, unani and homeopathy. The present study attempts to investigate the qualitative phytochemical constituents from extracts of the flower of Saraca asoca (Roxb.)Wilde. Tannin, flavonoids, carbohydrates, proteins, steroids have been found in different extracts of the flowers. Phytochemical as well as physicochemical analysis is useful to establish the authenticity of the crude drug form. A sensitive and reliable high performance thin layer chromatographic (HPTLC) method has been developed for qualitative determination of a pharmacologically important active constituent- gallic acid in the dried flowers and leaves of Saraca asoca (Roxb.)Wilde. The assay combines the separation of analytes on precoated silica gel 60 F 254 Keywords: Saraca asoca, Gallic acid, Flower, Leaf, HPTLC HPTLC plates as stationary phase and Toluene: Ethyl Acetate: Formic Acid: Methyl Alcohol (6:6:1.6:0.4 v/v/v/v) as mobile phase. Detection has been carried out densitometrically at λ =280 nm for gallic acid. This is the first report of occurrence of gallic acid in leaves of S. asoca. Further investigation will be carried out for the quantification of the test compound, gallic acid in Saraca leaf.
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PHYTOCONSTITUENTS AND HPTLC ANALYSIS IN SARACA ASOCA (ROXB.)WILDE
Research Article
JAYITA SAHA*, TANIYA MITRA*
Bethune College, Postgraduate and Undergraduate Department of Botany, 181 Bidhan Sarani, Kolkata 700006, West Bengal
Email: sumona.mukherjee13@gmail.com
, KAMALA GUPTA, SUMONA MUKHERJEE
Received: 5 Sep 2011, Revised and Accepted: 3 Oct 2011
ABSTRACT
Saraca asoca (Roxb.)Wilde, (Ashok) belonging to the family Caesalpiniaceae is a universal panacea in the ayurvedic medicine. This versatile plant
has anti-cancer, anti-menorrhagic, anti-oxytocic, antimicrobial activity and has extended uses in ayurveda, unani and homeopathy. The present
study attempts to investigate the qualitative phytochemical constituents from extracts of the flower of Saraca asoca (Roxb.)Wilde. Tannin,
flavonoids, carbohydrates, proteins, steroids have been found in different extracts of the flowers. Phytochemical as well as physicochemical analysis
is useful to establish the authenticity of the crude drug form. A sensitive and reliable high performance thin layer chromatographic (HPTLC) method
has been developed for qualitative determination of a pharmacologically important active constituent- gallic acid in the dried flowers and leaves of
Saraca asoca (Roxb.)Wilde. The assay combines the separation of analytes on precoated silica gel 60 F 254
Keywords: Saraca asoca, Gallic acid, Flower, Leaf, HPTLC
HPTLC plates as stationary phase and
Toluene: Ethyl Acetate: Formic Acid: Methyl Alcohol (6:6:1.6:0.4 v/v/v/v) as mobile phase. Detection has been carried out densitometrically at λ
=280 nm for gallic acid. This is the first report of occurrence of gallic acid in leaves of S. asoca. Further investigation will be carried out for the
quantification of the test compound, gallic acid in Saraca leaf.
INTRODUCTION
Medicinal herbs are moving from fringe to mainstream uses with a
great number of people seeking remedies and health approaches
free from side effects caused by synthetic chemicals. Ashoka is one
of the most ancient trees of India commonly known as an “ashok
briksh. Saraca asoca (Roxb.)Wilde or Saraca indica belongs to the
family Caesalpiniaceae. All the plant parts are considered to contain
medicinal properties. Leaves of Saraca asoca are known to contain
carbohydrates, proteins, tannins and saponins1 and shows
antibacterial activity1. Barks contain glycosides, steroids, saponins,
carbohydrates and tannins2. The flowers (Figure1) are also regarded
as medicinally important plant part and used as therapeutic agent in
treatment of diabetes, cancer and hemorrhagic dysentery, uterine
infections as menorrhagia and other types of uterine disorders. It is
also used in bleeding piles, bacillary dysentery.
Fig. 1: Flowers of Saraca asoca (Roxb.)Wilde.
Dried flower buds are reported to have antibacterial activity3.
Aqueous suspension of Saraca indica flower has antiulcer activity in
albino rats4. Saraca asoca bark and flowers exhibit antitumour
activity against DLA, S-180 and Ehrlich ascites carcinoma tumour
cell lines5. Larvicidal activity has also been recorded6. Chemo
preventive activity of flavonoid fraction of S. asoca is reported in
skin carcinogenesis7. During ‘ashoka-sasthi’ the flower buds are
taken orally by women. Though phytoconstituents have been
reported earlier in case of leaves and bark of the plant1,2, no detail
qualitative phytochemical analysis are found for flowers. The
present paper attempts to cover this lacuna by evaluating the
phytoconstituent standardization parameters in flowers that can
possibly be useful for identification of the drug in the dry form.
However, the presence of gallic acid, an antioxidant molecule, has
already been reported in Saraca asoca flower2, but there is no
reference about its presence in Saraca leaf. The detection and
qualitative assay has been developed by HPTLC method for the
standard gallic acid8
MATERIALS AND METHODS
. Simple HPTLC method is used to confirm the
presence of gallic acid in Saraca asoca flowers as well as leaves.
Collection and authentication of plant material
The flowers and leaves of Saraca asoca were collected in March
2011 from the campus of Bethune College, Bidhan Sarani, Kolkata,
and West Bengal, India. The species was authenticated by Professor
Gour Gopal Maity of University of Kalyani , West Bengal.
Preparation of extracts
Plant samples were washed with water and air-dried at room
temperature for 7 days, then oven-dried at 40ºC to remove the
residual moisture. The dried flowers and leaves were pulverized and
stored in air-tight container for future use. Four different solvents
were used for preliminary phytochemical screening - chloroform,
ethanol, methanol and distilled water. Equivalent amount of
powdered samples of flower were extracted with chloroform,
ethanol and methanol successively at room temperature for 3 days.
Water extraction was done in water bath at 60ºC. The four filtrates
were separately concentrated in water bath at 45ºC and evaporated
under reduced pressure and then the percent extract yield (%) was
calculated.
Phytochemical analysis
The four extracts obtained from the powdered flowers of Saraca
asoca were subjected to phytochemical tests to determine the
presence of active secondary metabolites using standard procedures
9
Physico-chemical parameter
.
Loss on Drying: 5 gm of the drug was weighed in an evaporating
dish. It was dried at 105°C for 3 hours and weighed again. The drying
and weighing was continued at intervals of one hour until difference
between two successive weights was not more than 0.25%.
1
International Journal of Pharmacy and Pharmaceutical Sciences
ISSN- 0975-1491 Vol 4, Suppl 1, 2012
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Saha et al.
Int J Pharm Pharm Sci, Vol 4, Suppl 1, 96-99
97
Ash Values: Ash values are useful in determination of crude drug in
the powder. The ash values represent the inorganic salt present in the
drug. The different ash values such as total ash, acid insoluble ash and
water soluble ash were determined from shade dried flower powder.
HPTLC analysis
Chemicals and standard gallic acid
Gallic acid was obtained from Titan Biotech Ltd. and Methanol,
Toluene, Ethyl Acetate, Formic Acid were used of analytical grade E-
Merck. Silica gel 60 F 254 precoated Thin Layer Chromatography
(TLC) aluminium plate was used of E-Merck.
Preparation of standard and sample solution
5 mg Gallic acid was dissolved in 3 ml of methanol. It was then
sonicated for 5 min and the final volume was made upto 5 ml with
the same solvent to obtain stock solution containing 1 mg/ml. Air
dried samples (0.5g) was extracted with 10 ml of methanol. Extracts
were concentrated, filtered and the final volume made upto 10 ml
with methanol prior to HPTLC analysis to get stock solution
containing 50 mg/ml.
Chromatographic conditions
Chromatography was performed on precoated silica gel 60 F 254
Plate was eluted in pre-saturated CAMAG twin trough glass tank
with the mobile phase Toluene: Ethyl Acetate: Formic Acid: Methyl
alcohol (6:6:1.6:0.4 v/v/v/v) to a distance of 86.2 mm at room
temperature. After drying, the spots were visualized under CAMAG
UV cabinet (254 and 280 nm). Then the plate was scanned using
CAMAG TLC scanner 3 equipped with WINCATS software (CAMAG).
The identification of gallic acid in methanolic solution of flower and
leaf of Saraca asoca was confirmed by superimposing the UV spectra
of samples and standards within the same retention factor (R
HPTLC plates (10.0 x 10.0 cm). Methanolic solutions of standard
compound (gallic acid) and samples of known concentrations were
applied to the plate positioned at 10 mm from the bottom and 19
mm from the side of the plate having 8 mm bandwidth using a
CAMAG Linomat 5 automated TLC applicator with the nitrogen flow
providing a delivery speed of 150 nl/s from the syringe.
Detection of Gallic acid
f
Preliminary phytochemical results indicate the presence or absence
of phytochemical-constituents in different extracts of the Saraca
flower (Table 1). Alkaloids were found to be absent in all the four
extracts. Carbohydrates, tannin, flavonoid, saponin, glycosides,
proteins and steroids were found to be present in methanol and
ethanol extracts. The chloroform extract contained only
carbohydrates whereas the water in addition to carbohydrates,
contain tannin, flavonoid, saponins, and steroids.
)
value.
RESULTS
Phytochemical and physicochemical analysis
The experimental yield of chloroform, ethanol, methanol and water
extracts of Saraca flower were found to be 1.80% 11.90%, 15.10%
and 22.00% respectively (Table 2). Water soluble extractive value
showed the presence of sugar, acids and inorganic compounds and
alcohol soluble extractive values determined the presence of polar
constituents. The physicochemical parameters total ash, acid
insoluble ash and water soluble ash value were found to be 3.00%,
2.02% and 1.12% respectively (Table 3). Total ash value percentage
showed the amount of mineral and earthy material present in the
plant sample. The amount of acid insoluble siliceous matter present
in the plant sample was 2.02% (Table 3).
Table 1: Table shows preliminary phytochemical screening of Saraca asoca flower extracts
Phytoconstituents Test/ Reagent Chloroform extract Ethanol extract Methanol extract Aqueous extract
Alkaloids
Dragendorff’s test
-
-
-
-
Hager’s test
-
-
-
-
Wagner’s test
-
-
-
-
Proteins
Biuret test
-
+
+
-
Ninhydrin’s test
-
+
+
-
Millon’s test
-
+
+
-
Tannins
Lead Acetate
-
+
+
+
Potassium dichromate
-
+
+
+
Ferric Chloride
-
+
+
+
Steroids
Salkowski test
-
+
+
+
Carbohydrates
Fehing’s test
+
+
+
+
Benedict’s test
+
+
+
+
Barfoed’s test
+
+
+
+
Flavonoids
Sodium hydroxide test
+
+
Conc. H2SO 4
-
test
+
+
+
Glycosides
Borntrager’s test
-
+
+
-
Keller-Killani test
-
+
+
-
Saponins
Foam test
-
+
+
+
(+)Present (-)Absent
Table 2: Extractive values of Saraca asoca flowers
Types of solvents % w/w
Chloroform
1.80
Ethanol
11.90
Methanol
15.10
Water
22.00
Table 3: Ash values and loss on drying of Saraca asoca flowers
Saha et al.
Int J Pharm Pharm Sci, Vol 4, Suppl 1, 96-99
98
Total ash
3.00
Acid insoluble ash
2.02
Water soluble ash
1.12
Loss on Drying
12.90
HPTLC analysis
Photograph of chromatograms of the standard Gallic acid at 280 nm
obtained in the methanolic extract of dried flower extract and leaf
extract of Saraca asoca are given in Figure 2. The Gallic acid bands in
sample chromatogram of Saraca sp. are identified and confirmed by
comparing the chromatogram obtained from the reference standard
solution (Figures 3- 5) and by comparing retention factor(Rf) of
Gallic acid from sample and standard solution. The Rf value of
standard gallic acid is 0.44, whereas the Rf value of methanolic
extract of the flower and leaf of Saraca asoca is 0.43 which almost
coincides with standard Rf
value of gallic acid (Figure 2).
Fig. 2: Photograph of chromatograms obtained at 280nm from
standard Gallic acid (1)Flower extract(2) and Leaf extract (3) of
Saraca asoca
Fig. 3: Chromatogram of standard Gallic acid
Fig. 4: Chromatogram of Gallic Acid in the methanolic extract of
flower of Saraca asoca
Fig. 5: Chromatogram of Gallic Acid in the methanolic extract of
leaf of Saraca asoca
DISCUSSION
Preliminary phytochemical screening revealed the presence of
tannins, proteins, steroids, glycosides, carbohydrates, saponins,
flavonoids in different extracts of the flower of Saraca asoca. These
results show that flowers of Saraca asoca contain a number of
chemical ingredients, which may be responsible for the various
pharmacological actions although their specific roles remain to be
investigated. It has been observed that most active principles
present in the flowers are flavonoid, steroids, tannins and
glycosides. These phytoconstituents may be responsible for various
pharmacological actions of this plant part, like antibacterial,
antiulcer, anticancer, larvicidal and chemo protective activities
3,4,5,6,7. Methanolic extracts of flower and leaves confirmed the
presence of gallic acid using HPTLC assay. This is the first report of
the presence of gallic acid in Saraca asoca leaf. The presence of gallic
acid in leaf is very important since flower is only seasonal, while leaf
is available all year round. Hence, the amount of gallic acid in Saraca
asoca leaf can be quantified further for proper utilization of this age
old plant. The physicochemical evaluation of this plant is an
essential parameter for the detection of adulterant and improper
handling of drugs. The present work can serve as a valuable source
of information and provide appropriate standards to establish the
quality of this plant material in future study or application.
Saha et al.
Int J Pharm Pharm Sci, Vol 4, Suppl 1, 96-99
99
ACKNOWLEDGEMENT
The authors acknowledge Ramakrishna Mission Quality Testing
Laboratory (QTL), Vivekananda University, Narendrapur, for
providing facilities for carrying out this research work. The authors
gratefully acknowledge Dr. Chhanda Mandal for her guidance and
Prof. Gour Gopal Maity for identification of plant material. The
financial help from West Bengal Biodiversity Board is duely
1. Pradhan P, Joseph L, George M, Kaushik N, Chulet R.
Pharmacognostic, Phytochemical & Quantitative Investigation
of Saraca asoca leaves. Journal of Pharmacy Research. 2010;
3(1): 776-780.
acknowledged.
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... 21 Parameters like total ash, acid insoluble ash, and water-soluble ash were studied up on dried flower powders. 55 Parameters like foreign matter, water-soluble extractive, alcoholsoluble extractive, water-soluble ash, acid-insoluble ash, total ash value, foaming index, moisture content, and crude fiber content evaluated on leaves, can be further developed for identification of the original drug material. 29 ...
... Methanol and ethanol flower extract was found to contain carbohydrates, tannins, flavonoids, glycosides, saponin, proteins, and steroids. 55 Petroleum benzene, acetone, water, ethanol, and diethyl ether extracts of flower, leaves, and bark revealed the presence of saponins, steroids, phenols, carbohydrates, glycosides, phytosterols, and tannins. 56,57 The presence of terpenoids and phlobatannins has also been reported in Ashoka leaves. ...
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This work was designed to estimate the protective effect of Saraca indica L. leaves ethanolic exract against γ‐irradiation induced renal damage in rats. Phytochemical examinations of S. indica L. leaves extract resulted in the separation of three flavanone glycosides: Astilibin (1), Neoastilbin (2), and Eriodictyol‐7‐O‐α‐l‐rhamnopyranoside (3); two flavonols: Quercetin (4) and Quercetin‐3‐O‐α‐l‐arabinopyranosyl‐(1′′′‐6′′)‐O‐β‐D‐galactopyranoside (5) in addition of Gallic acid (6) and methyl gallate (7). Their structures elucidated by chemical evidences and spectroscopic analysis (1 and 2D‐NMR, ‐ESI‐MS, UV). Female rats were used and classified into: control, Ext (200 mg/kg body wt/day orally for 7 days), IRR (8Gy), Ext + IRR, and Sily+IRR groups (received silymarin 50 mg/kg b.wt orally as reference drug). Results showed that S. indica L. leaves extract ameliorated the kidney function tests, hs‐CRP, IL‐1β, ACE, TNF‐α, GSH, and MDA as well as, decreased the histopathological changes of kidney. In conclusion, S. indica L. leaves extract had a renoprotective activity against irradiation induced renal injury due to its flavononid contents.
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Ayurveda is a traditional system of medicine in which herbal therapies were used systematically. Ashoka i.e. Saraca indica Linn. belonging to Caesalpinaceae subfamily of the Legume is one of the indigenous plants with lots of traditional significance. Ashoka means "without sorrow, a reference to reputation of its bark for keeping a woman healthy and youthful. It has been mentioned in some of the oldest Indian Literature apart from Ayurveda. It is well described in
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An HPTLC method was developed for the estimation of gallic acid rutin and quercetin from aqueous extract of Terminalia chebula, precoated silica gel GF 254 as stationary phase and mobile phase for tannins toluene: acetone: glacial acetic acid (3:1:2 v/v/v/v/v) and mobile phase for rutin and quercetin, ethyl acetate: dichloromethane: formic acid: glacial acetic acid: water (10:2.5:1:1:0.1, v/v/v/v/v). Detection and quantification were performed densitometrically at λ = 254 for gallic acid and 366 nm for rutin and quercetin. The R f values of gallic acid, rutin and quercetin are 0.30, 0.13 and 0.93 respectively. The total peak areas of the standards (gallic acid, rutin and quercetin) and the corresponding peak areas of extracts were compared and the Gallic acid, rutin and quercetin content was estimated to be 8.380, 0.170 and 0.10331 % w/w.
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Screening of natural products for mosquito larvicidal activity against three major mosquito vectors Aedes aegypti, Culex quinquefasciatus, and Anopheles stephensi resulted in the identification of three potential plant extracts viz., Saraca indica/asoca, Nyctanthes arbor-tristis, and Clitoria ternatea for mosquito larval control. In the case of S. indica/asoca, the petroleum ether extract of the leaves and the chloroform extract of the bark were effective against the larvae of C. quinquefasciatus with respective LC(50) values 228.9 and 291.5 ppm. The LC(50) values of chloroform extract of N. arbor-tristis leaves were 303.2, 518.2, and 420.2 ppm against A. aegypti, A. stephensi, and C. quinquefasciatus, respectively. The methanol and chloroform extracts of flowers of N. arbor-tristis showed larvicidal activity against larvae of A. stephensi with the respective LC(50) values of 244.4 and 747.7 ppm. Among the methanol extracts of C. ternatea leaves, roots, flowers, and seeds, the seed extract was effective against the larvae of all the three species with LC(50) values 65.2, 154.5, and 54.4 ppm, respectively, for A. stephensi, A. aegypti, and C. quinquefasciatus. Among the three plant species studied for mosquito larvicidal activity, C. ternatea was showing the most promising mosquito larvicidal activity. The phytochemical analysis of the promising methanolic extract of the seed extract was positive for carbohydrates, saponins, terpenoids, tannins, and proteins. In conclusion, bioassay-guided fractionation of effective extracts may result in identification of a useful molecule for the control of mosquito vectors.
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Ashoka is the most ancient tree of India, generally known as a "ashok briksh", botanist known as a Saraca asoca (Roxb.), De.wild or Saraca indica belonging family Caesalpinaceae. Medicinal herbs are moving from fringe to mainstream use with a great number of people seeking remedies and health approaches free from side effects caused by synthetic chemicals. Saraca asoca is reported to contain glycoside, flavanoids, tannins and saponins. It is used as spasmogenic, oxytocic, uterotonic, anti-bacterial, anti-implantation, anti-tumour, anti-progestational, anti-estrogenic activity against menorrhagia and anti-cancer. This review contains the Pharmacognostical account of various parts of plant, Phytochemical constituent and different reported pharmacological activity.
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Saracin, a seed integument lectin from Saraca indica is highly specific for binding N-acetyl-neuraminyl-N-acetyllactosamine [Neu5Ac-α-(2-6)/(2-3)-d-Gal-β-(1-4)-d-GlcNAc]. This lectin has been found to be mitogenic for human lymphocytes, and this mitogenic activity could be inhibited in presence of fetuin. Further, treatment with saracin could induce secretion of IL-2 in a culture of resting human peripheral blood mononuclear cells (PBMC) after 48 h. Saracin has a higher affinity for the CD8+ than CD4+ T cells as revealed by FACS analysis. Agarose gel electrophoresis of DNA isolated from lymphocytes cultured under different conditions has shown that this lectin could induce apoptosis in activated T-lymphocytes, as also confirmed by flow cytometric studies. Phenotypic analysis of the apoptotic cells reveals that they belong to CD8+ T cells lineage. Four surface glycoproteins of PBMC have been found to interact with saracin in a trisaccharide [Neu5Ac-α-(2-6)/(2-3)-d-Gal-β-(1-4)-d-GlcNAc]-sequence specific manner. Saracin seems to be an interesting immunomodulator for the mammalian immune system.
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Saraca asoca family (Caesalpiniaceae) commonly known as Ashoka briksh. According to our traditional system of medicine, bark of Saraca asoca has tremendous therapeutic effects in menstrual problems. Establishment of Pharmacognostic profile of the leaves will assist in standardization which can guarantee quality, purity and identification of samples. In the present study we describe Pharmacognostical feature, including macroscopy, microscopy and analytical profile and to investigate the phytochemical and quantitative determination of phytoconstituents from the leaves of Saraca asoca. These observations would be of immense value in the botanical identification and standardization of the drug in crude form. This study would help distinguish the drug from its adultered species.
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Saraca asoka (Family - Caesalpiniaceae) has been widely used in the Ayurvedic (traditional Indian) system of medicine especially due to its wound healing property. The present study investigated the chemopreventive property of flavonoids from the flowers of Saraca asoka on 7,12 dimethyl benz(a)anthracene (DMBA) induced skin cancer in mice models. A single topical application of DMBA (100 microg/50 microL of acetone) followed after 2 weeks by three times a week treatment with croton oil (1% in acetone), for 20 weeks resulted in tumor induction. The topical application of the flavonoid fraction of S. asoka (FF S. asoka), 30 min prior to the application of croton oil thrice weekly for 20 weeks, caused a significant reduction in the number of tumors per mouse and the percentage of tumor-bearing mice. Also the latency period for the appearance of the first tumor was delayed by S. asoka pretreatment. In the flavonoid fraction (5 mg and 10 mg/kg body weight) treated animals, the levels of biochemical markers - rhodanese, myeloperoxidase, beta-D-glucuronidase, sialic acid, hexokinase and caspase 3 were significantly restored to near normal levels. These findings suggest the chemopreventive activity of flavonoids from S. asoka on two stage skin carcinogenesis. Histological data also support the chemopreventive potential of S. asoka.
Saraca asoca (Ashoka)
  • P Pradhan
  • L Joseph
  • V Gupta
  • R Chulet
  • H Arya
  • R Verma
  • A Bajpai
Pradhan P, Joseph L, Gupta V, Chulet R, Arya H, Verma R, Bajpai A. Saraca asoca (Ashoka). A Review Journal of Chemical and Pharmaceutical Research. 2009; 1 (1): 62-71.
  • W C Evans
  • G E Trease
Evans WC, Trease GE. Pharmacognosy. London. WB Saunders. 1996; 14:119-159.