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Table 1: Phytochemical screening of the BCAE
Phytoconstituents Phytochemical test Inference BCAE
Carbohydrates Molisch’s test Formation of violet ring at junction +
Proteins Biuret test Appearance of violet color -
Xanthoproteic test Formation of white precipitate
Amino acids Ninhydrin test Appearance of Purple color -
Triterpenoid sterols Salkowski reaction Appearance of red color in chloroform layer +
while greenish yellow in acid layer
Fats, oils and volatile oils Solubility test Solubility in water, ether, benzene and -
chloroform
Fats and oils Saponification test Formation of soap -
Glycosides Keller Killiani test Formation of reddish brown colour at junction +
Flavonoids Shinoda test Formation of reddish to pink color +
Alkaloids Dragendroff’s test Formation of orange colour precipitate +
Wagner’s test Formation of reddish brown precipitate
Phenolic compounds Lead acetate test Formation of white precipitate +
and tannins Test with FeCl Appearance of bluish black color
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+: present; -: absent; BCAE: aqueous extract of Bombax ceiba
guidelines prescribed by The Committee for the Purpose Assessment of liver function test and hepatic
of Control and Supervision of Experiments on Animals and damage
the use of animals was approved by the Institutional Animal On the eigth day of the experiment, blood was withdrawn
Ethics Committee of the Institute (Proposal No. CRI-GWL/ by micro-capillary technique from the retro-orbital plexus
IAEC/2010/08). under light ether anesthesia. This technique is used with
recovery in experimental circumstances and this method is
Acute toxicity study also called periorbital, posterior-orbital and orbital venous
Healthy Wistar rats, starved overnight, were subjected to plexus bleeding. Briefly, a capillary is inserted into the medial
acute toxicity studies to determine non-observable adverse canthus of the eye (30 degree angle to the nose) with a
effect dose level (NOAEL) by acute toxic class method of slight thumb pressure to puncture the tissue and enter the
oral toxicity as per Organization for Economic Co-operation plexus/sinus. Once the plexus is punctured, blood will come
and Development 423 guidelines. The rats (n = 3) were through the capillary tube which was collected in 1.5 mL
[17]
administered BCAE in the limit test dose of 2000 mg/kg and Eppendorff tubes from the plexus. The capillary tube is then
observed continuously for behavioral, neurological, and gently removed and wiped with sterile cotton. Bleeding can
autonomic profiles for 2 h, and after a period of 24, 72 h and be stopped by applying gentle finger pressure. Blood was
[20]
thereafter up to 14 days for any lethality, moribund state, or centrifuged at 3,000 g to obtain plasma, which was used
death. The limit test was repeated in another group of rats to assess liver function parameters (GOT, GPT, ALP, T, [25]
[24]
[23]
(n = 3) for confirmation and approximate LD determination.
[26]
50 total protein, albumin and TG) using semi-autoanalyser
Experimental induction of hepatotoxicity (Microlab 300, Merck Specialities Pvt. Ltd. New Delhi).
Hepatotoxicity was induced in Wistar rats by intraperitoneal Histological studies
(i.p.) administration of CCl in olive oil in the ratio of 1:1 at
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the dose of 1 mL/kg for two continuous days as described After the withdrawal of blood, the animal was sacrificed by
previously with modifications. [18,19] After 48 h of the last cervical dislocation. Abdomen was cut opened and aorta
dose of CCl , blood was withdrawn from retro-orbital plexus was cut to washout the blood from tissues. The liver was
4
by capillary puncture method. Plasma was separated dissected out. A piece of liver was fixed in 10% v/v neutral
[20]
and analyzed for the various biochemical markers of buffered formalin. Serial sections (4-5 μm thick) of the
hepatotoxicity and hepatic damage. paraffin-embedded tissue blocks were cut with a Microm
HM 360 microtome and processed for hematoxylin and eosin
Grouping and treatments (HE), Masson’s trichrome (Accustain Trichrome Stains, Sigma-
The rats were divided into five groups (n = 5 each). Group I Aldrich Inc, USA). Staining was done as per manufacturer’s
received only olive oil (1 mL/kg, i.p.), and remaining groups protocol. The sections were studied under microscope.
(group II, III, IV and V) received 1 mL/kg, i.p. CCl in olive oil
4
for two continuous days. While group II (control) received Assessment of antioxidant activity
the vehicle of the extract (5 mL/kg, distilled water, orally), Quantitative estimation of antioxidant phytochemicals
group III and IV received BCAE (250 and 500 mg/kg orally, The total phenolic content of the extracts was determined
[27]
respectively). Group V received silymarin suspension (25 spectrometrically and expressed as milligrams of tannic
mg/kg, orally), a known antioxidant and hepatoprotective acid equivalents (TAE) per gram of extract. Total flavonoid
agent. [21,22] The vehicle/drugs were administered daily content was measured by aluminum chloride colorimetric
orally for seven days and CCl administration was done on assay and expressed as milligrams of quercetin equivalent
[28]
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the 5th and 6th day of vehicle/drug treatments. per gram of extract.
146 Hepatoma Research | Volume 2 | June 1, 2016