Journal of Herbal Medicine and Toxicology 4 (2) 101-106 (2010)

ISSN : 0973-4643

Original Article

HEPATOPROTECTIVE ACTIVITY OF SOME PLANTS EXTRACT

AGAINST PARACETAMOL INDUCED HEPATOTOXICITY IN RATS
Simon Rama Parmar1, Patel Hitesh Vashrambhai2, Kiran Kalia2
1

Department of Biochemistry, M.B. Patel Sciences Collage, Anand-388 001; 2Laboratory of Biochemistry,
BRD School of Biosciences, Sardar Patel University, V.V.Nagar 388 120, Gujarat, India
Correspondence Author: Dr. (Mrs.) Kiran Kalia, Professor in Biochemistry, E-Mail:
kirankalia_in@yahoo.com, cell- +91 2692 234412-Ex 302, 09824335881 Fax: +91 2692 231042
Received-12th April,2010, Accepted- 26th April 2010

Abstrct: Paracetamol (PCM) is widely used as analgesic and antipyretic drug, but at
high dose it leads to undesirable side effects, such as hepatotoxicity. The present study
demonstrates the comparative hepatoprotective and antioxidative activity of Phyllanthus
niruri, Maytenus emarginata, Eclipta alba, Aloe Vera, Solanum indicum and Aegle
marmelos against paracetamol induced toxicity. Paracetamol induce hepatotoxicity
was evaluated by an increase (P<0.05) in serum AST, ALT, ALP activity and bilirubin
level accompanied by significant decrease (P<0.05) in albumin level. Paracetamol
hepatotoxicity was manifested by an increase (P<0.05) lipid peroxidation, depletion
of reduced glutathione (GSH) and catalase activity in liver tissue. Co-administration
of all above aqueous plants extract [22.0 g% (w/v)] protects the paracetamol induced
lipid peroxidation, restored altered serum marker enzymes and antioxidant level
towards near normal. The results indicate hepatoprotective and antioxidative activity
of all studied plants extract against paracetamol induced toxicity. Phyllanthus niruri
and Maytenus emarginata exhibits maximum hepatoprotective activity against
paracetamol induced toxicity as compared to the other studied plants.
Key Words: paracetamol, hepatoprotective, antioxidative activity, medicinal plants
INTRODUCTION
Liver diseases have become one of the major causes
of morbidity and mortality all over world. From
among, drug induced liver injury (DILI) is one of the
most common causative factor that poses a major
clinical and regulatory challenge [1]. The
manifestations of drug-induced hepatotoxicity are
highly variable, ranging from asymptomatic elevation
of liver enzymes to fulminant hepatic failure.
Paracetamol (PCM) also known as Acetaminophen,
taken in overdose can cause severe hepatotoxicity
and nephrotoxicity [2]. PCM is activated and
converted by cytochrome P450 enzymes to toxic
metabolite NAPQI (N-acetyl-p-benzoquinoneimine)
that causes oxidative stress and glutathione (GSH)
depletion [2, 3]. In spite of tremendous advances in
modem medicine, there are hardly any reliable drugs
that protect the liver from damage and/or help in

regeneration of hepatic cell. Many active plant

extracts are frequently utilized to treat a wide variety
of clinical diseases including liver disease [4].
Therefore, searching for effective and safe drugs for
liver disorders are continues to be an area of interest.
Aegle mameloes, belongs to family Rutaceae is
commonly known as beal. It exhibits contraceptive,
hypoglycemic activity, [5] cardio protective effects
and anti-hyperlipidaemic activity [6]. Aloe vera
(family-Liliaceae) has been reported to have
antifungal activity, hypoglycemic activity, antiinflammatory, anticancer, immunomodulatory, gastroprotective and anti-hepatotoxic properties [7]. Eclipta
alba (family-Asteraceae) commonly known as
Bringraj, has been shown to posses antiinflammatory, anti-hypertensive, anti-hepatotoxic,
immunomodulatory, anthelmintic, expectorant,

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antipyretic, anti-asthmatic and deobstruent properties
[8, 9]. Phyllanthus niruri, belongs to family
Euphorbiaceae, commonly known as bhoya amali.
The plant has demonstrated antihepatotoxic,
hypotensive, antispasmodic, antibacterial, diuretic,
hypoglycemic activities [10] and shows inhibitory
effect on the formation of kideny stone [11]. Solanum
indicum belongs to Solanaceae family, exhibits
antihepatotoxic activity and is widely used in loss of
appetite, abdominal pain, distaste [12]. Maytenus
emerginata is (familyCelastraceae) commonly
known as viklo. It exhibits antioxidant and cytotoxic
activity [13].

Experimental protocol: Animals were divided into eight

groups of six rats each and treated orally as below
for 15 days.

The present study was conducted to compare antihepatotoxic and antioxidant potential of above
mentioned medicinal plants extract against
paracetamol-induced toxicity in rats.

Group-V (P3): Paracetamol as in group-II + E. alba

extract 440mg/kg b. wt.

MATERIALS AND METHODS

Group-VII (P5): Paracetamol as in group-II + S.

indicum extract 440mg/kg b. wt.

Drugs and Chemicals: Sulphosalicylic acid, Dithiobis2-nitrobenzoic acid (DTNB), 2-trichlorobarbituric acid
(TBA) was procured from Hi-Media, Bombay, India.
Paracetamol drug (Sun Pharmaceuticals Ltd)
purchased from local medical store. All reagents
procured were analytical grade.
Plant Material: Fresh leaves of P. niruri, M.
emarginata, E. alba, A. Vera, S. indicum and A.
marmelos were collected from Anand Agriculture
University (AAU), Anand (Gujarat). Our
departmental Taxonomist has identified the plants.
They were cut into small pieces and air dried indoors
under subdued light and with good ventilation. They
were then crushed into a course powder using a
laboratory homogenizer, which was used as a plant
material to make 22.0 g % (w/v) crude homogenate
in distilled water, filtered through double layer of muslin
cloth and refiltered using Whatmans filter paper.
Animals: Male Wistar rats weighing 200 20g were
used for this study. The animals were kept in
polypropylene cages and maintained at 25 5C under
12 h light/dark cycle. The animals were allowed free
access standard pellet diet (Pranav Agro Industries,
Pune) and water ad libitum. The animal experiment
was approved by research committee of Sardar Patel
University, V.V.Nagar and performed according to
the guidelines laid by Institutional Animal Ethical
Committee (IAEC).

Group-I: served as normal control received distilled

water.
Group-II: as paracetamol control, administered with
paracetamol 500mg/kg body wt. in distilled water.
Group-III (P1): Paracetamol as in group-II + P. niruri
extract 440 mg/kg b. wt.
Group-IV (P2): Paracetamol as in group-II + M.
emerginata extract 440 mg/kg b. wt.

Group-VI (P4): Paracetamol as in group-II + A. vera

extract 440mg/kg b. wt.

Group-VIII (P6): Paracetamol as in group-II + A.

marmelos extract 440mg/kg b. wt. Animals were
sacrificed under light ether anesthesia 24-h after the
last dose. Blood was collected by cardiac puncture in
plain tubes and liver was removed, rinsed in cold saline,
blotted with filter paper and weighted. 10% (w/v) liver
homogenate was prepared in 0.25M sucrose solution
and centrifuged at 7000 rpm for 10 min at 4C.
Supernatant was used for various biochemical assays.
Protein concentration was estimated according to the
method of Lowry et al., [14] using bovine serum
albumin (BSA) as a standard.
Biochemical Assays
Serum marker enzymes of liver function: Serum was
separated by centrifugation at 3000 rpm at 4C for
10min and used for measurement of various
biochemical markers like aspartate and alanine
aminotransferase (AST and ALT) activities, alkaline
phosphatase (ALP) activity, total bilirubin and total
albumin using commercially available kits. (Crest Bio
Systems, A division of Coral Clinical Systems, Verna,
Goa-403 722, India)
Oxidative stress markers in liver tissue: Lipid
peroxidation -Lipid peroxidation was measured by the
method of Onkawa et al., [15]. Liver homogenate
was incubated with 8.1% SDS (w/v) for 10 min

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Parmar et al.
followed by addition of 20% acetic acid (pH 3.5).
Reaction mixture was incubated with 0.6% TBA (w/
v) for 1 h at 100oC. Pink colored complex was
extracted in butanol: pyridine [15:1 (v/v)] solution and
read at 532 nm. The amount of TBARS was
calculated using a molar extinction coefficient of
1.56105 M/cm.
Catalase activity: The catalase activity was measured
according to method of Shinha et al., [16]. 0.1ml of
liver homogenate was mixed with 1.0 ml of 0.01M
phosphate buffer (pH 7.4), and incubated with 0.4 ml
of 0.2M H2O2 at 37C accurately for 1.0 min and
reaction was stopped with 2.0 ml of 5% potassium
dichromate (1:3 with glacial acetic acid). Further the
samples were incubated in boiling water bath for 15
min. Tubes were centrifuged at 5000 rpm for 15 min
and supernatant was used to quantify the amount of
H2O2 to calculate catalase activity at 570 nm. One
unit represents 1.0mole of H2O2 consumed/min/mg
protein.
Reduced Glutathione: Hepatic reduced glutathione
(GSH) level was determined by the method of Ellman
modified by Jollow et al. [17]. 0.5 ml 10%
sulphosalicylic acid was added to mixture of 0.4 ml
homogenate and 0.6 ml of distilled water as protein
precipitant. 0.5 ml of supernatant was mixed with
the reaction mixture of 4.5 ml of 0.5M Tris-buffer
and 0.5 ml of 10mM DTNB and the absorbance was
measured immediately at 412 nm .The GSH contents
were calculated using GSH as standard and expressed
as mM/g tissue.
Statistical analysis: The values were expressed as
meanSD. Statistical analysis and comparison
between the groups was performed by one way
analysis of variance (ANOVA) using SPSS version
10.0, followed by LSD (Least Significance
Difference) test. Difference between unexposed and
exposed (with or without treatment) with a p-value <
0.05 was considered significant.
RESULTS
Serum biochemical parameters: Serum biochemical
parameters in the control and various experimental
groups are depicted in Table 1. Administration of
paracetamol to rats by oral route caused liver damage
as indicated by a significant increase in serum enzyme
ALT, AST, ALP activity and bilirubin level (by 183%,

136.8%, 107.3% and 85.2% respectively) compared

to control rats. Elevated levels of these enzymes are
indicative of cellular leakage and loss of functional
integrity of cell membrane in liver. Co-administration
of rats with aqueous extract of E. alba, A. Vera, S.
indicum remarkably (P < 0.05) restored paracetamol
induced elevated serum level of ALT (31%, 51% and
52%), AST (37%, 59% and 51%) and ALP (59%,
36% and 78%) towards normal value respectively. P.
niruri, M. emarginata and A. marmelos restore ALT
(69%, 73% and 67%), AST (68%, 81% and 67%)
and bilirubin level (84%, 88% and 95%) maximum
towards normal value respectively. (Table 1) ALP
activity was restored 100% by co-treatment with P.
niruri, M. emarginata and A. marmelos restore on
paracetamol induced toxicity. The obtained results
indicated a high degree of protection against the
hepatotoxic effect of paracetamol. The serum albumin
levels were significantly (P < 0.05) declined (54.82%)
in paracetamol treated group. Co-treatment with the
aqueous extract of P. niruri, M. emarginata, E. alba,
A. Vera, S. indicum and A. marmelos could significant
(P<0.05) restored serum albumin level by 46%, 64%,
51%, 33%, 25% and 21% respectively towards their
control values depicted in Table 1.
Oxidative stress markers in liver tissue: The levels of
TBARS as an index of lipid peroxidation, a
degradative process of membranous lipids, in liver
tissue of paracetamol treated rats were significantly
(P < 0.05) elevated (by 51.86%) when compared to
control animals. Lipid peroxidation level was restored
100% towards their normal value by treatment with
P. niruri and M. emarginata extract on paracetamol
induced toxicity. The increased TBARS level in
paracetamol treated group was significantly restored
by 47%, 51%, 63% and 83% on co-administration of
aqueous extract of E. alba, A. Vera, S. indicum and
A. marmelos respectively indicate prevention of
accumulation of lipid peroxidation. (Fig. 3)
Phyllanthus niruri and Maytenus emarginata
extract proved to be the best among all the other
extracts in protecting liver from peroxidative injury.
In the present study, we have observed that reduced
GSH level was depleted significantly (P < 0.05, by
79.4%) in the liver tissue of paracetamol treated rats
compared to control group. Co-treatment with the
aqueous extract of E. alba, A. Vera and S. indicum
had significantly improved level of GSH (restore by
48%, 36% and 42% respectively towards their control

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Table 1 : Effect of P. niruri (P1), M. emerginata (P2), E. alba (P3), A. vera (P4), S. indicum (P5) and
A. marmelos (P6) on serum marker enzymes (AST, ALT and ALP), bilirubin and albumin
on paracetamol induced hepatotoxicity in rats
Groups

Bilirubin (mg/dl)

Total-Albumin (g/dl)

ALT (U/L)

AST (U/L)

ALP (U/L)

Control

1.150.13

4.870.11

22.93 6.21

28.90 3.51

155.65 20.00

Para

2.130.62*

2.20.60*

64.99 4.00 *

68.49 3.92 *

322.11 25.82 *

P1

1.260.63
(88%)

3.610.15*
(46%)

35.75 3.75*
(69%)

41.39 3.32*
(68%)

143.73 18.92
(100%)

P2

1.300.16
(84%)

4.040.08*
(64%)

34.22 4.78*
(73%)

36.13 6.24
(81%)

125.42 22.62
(100%)

P3

1.190.03
(95%)

3.730.09*
(51%)

51.59 3.80*
(31%)

53.46 6.52*
(37%)

223.04 19.41*
(59%)

P4

1.210.63
(93%)

2.600.21*
(33%)

43.12 2.11*
(51%)

45.01 3.64*
(59%)

261.79 30.53*
(36%)

P5

1.680.31*
(45%)

2.750.15*
(25%)

42.73 1.55*
(52%)

48. 03 1.72*
(51%)

190.75 21.22*
(78%)

P6

1.190.12
(95%)

3.020.21*
(21%)

36.75 6.85*
(67%)

41.67 3.05*
(67%)

153.30 25.60
(100%)

Values are expressed as mean S.D. for six rats in each group. Value in bracket represents percentage restoration of
the activity to normal values

P < 0.05 denote value significantly different from control

P<0.05 experimental groups compared with paracetamol group

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Parmar et al.
value) in liver tissue. P. niruri, M. emarginata and
A. marmelos treated group shows maximum
restoration (72%, 70% and 66% respectively) of GSH
level compared to all other extracts on paracetamol
toxicity. Catalase activity was significantly (P < 0.05)
decline (by 47.5%) in paracetamol treated group
compared to the control. Co-treatment with the
aqueous extract of P. niruri, M. emarginata, E. alba,
A. Vera, S. indicum and A. marmelos remarkably
restore (37%, 31%, 14%, 22%, 40% and 43%
respectively) catalase activity towards their normal
level. Phyllanthus niruri and S. indicum and A.
marmelos provided more pronounced beneficial
effects to prevent decrease catalase activity on
paracetamol induced toxicity than the other plants
extract.
DISCUSSION
Paracetamol is a common analgesic and antipyretic
drug. Several studies have demonstrated the induction
of hepatocellular damage or necrosis by
acetaminophen higher doses in experimental animals
and humans [2]. For screening of hepatoprotective
agents, paracetamol-induced hepatotoxicity has been
used as a reliable method. Paracetamol is metabolized
primarily in the liver and eliminated by conjugation
with sulfate and glucuronide, and then excreted by
the kidney. Moreover, paracetamol hepatotoxicity has
been attributed to the formation of toxic metabolites,
when a part of paracetamol is activated by hepatic
cytochrome P-450 to a highly reactive metabolite Nacetyl-p-benzoquinoneimine (NAPQI) [18]. Toxic
metabolites (N-acetyl-p-benzoquineimine) can
alkylate and oxidise intracellular GSH, which results
in liver GSH depletion subsequently leads to increased
lipid peroxidation by abstracting hydrogen from a
polyunsaturated fatty acid and ultimately, liver damage
due to higher doses of paracetamol. [19,20] Reactive
metabolites can exert initial cell stress through a wide
range of mechanisms including depletion of glutathione
(GSH) or binding to enzymes, lipids, nucleic acids and
other cell structures [21].
AST predominantly found in mitochondria of
hepatocytes. ALT is more specific to liver, and thus
is a better parameter for detecting liver injury. Serum
ALP and bilirubin is also associated with liver cell
damage. The ALT, AST and ALP activity and serum
bilirubin level are largely used as most common

biochemical markers to evaluate liver injury [22, 23].

Administration of paracetamol caused a significant
elevation of enzymes level such as AST, ALT, ALP
and bilirubin level has been attributed to the damage
structural integrity of liver, because they are
cytoplasmic in location and released into circulation
after cellular damages indicating development of
hepatotoxicity [24, 25]. The co administrations of all
examined plants extract have prevented the increased
serum marker enzymes AST, ALT, ALP level and
bilirubin level. This is in agreement with the commonly
accepted view that serum levels of AST, ALT and
ALP return to normal with the healing of hepatic
parenchyma and the regeneration of hepatocytes [26].
Reduction of serum albumin in paracetamol treated
group may be due to formation of protein adduct.
Toxic metabolites NAPQI leads to covalent
modification of cellular target protein, cell death and
organ damage [27]. Among six studied plants, P.
niruri and M. emerginata exhibits the excellent
hepatoprotective properties as indicated by maximum
prevention of increased serum biochemical parameters
on paracetamol induced toxicity.
Catalase converts harmful hydrogen peroxide into
water and oxygen and protects the tissues from highly
reactive hydroxyl radicals [28]. The reduction in the
activity of this enzyme may results in number of
deleterious effect due to accumulation of highly toxic
metabolites and hydrogen peroxide on paracetamol
administration, which can induce oxidative stress in
the cells [21]. Co-administration of P. niruri, M.
emerginata, E. alba, A. vera, S. indicum and A.
marmelos increases the activities of catalase in
animals to prevent the accumulation of excessive free
radicals and protects the liver from paracetamol
intoxication. The increase in TBARS level in liver
induced by paracetamol suggests enhanced lipid
peroxidation leading to tissue damage and failure of
antioxidant defense mechanism. A massive decrease in
lipid peroxidation in liver tissue of plant extract treated
groups indicates that all studied plants possess antioxidative properties [29]. GSH removes free radical
species such as hydrogen peroxide, superoxide radicals
and maintains membrane protein thiols. The GSH
depletion in hepatic mitochondria is considered the most
important mechanism in the paracetamol induced
hepatotoxicity. Reduced GSH level was depleted in
paracetamol treated group may be due to conjugation
of GSH with NAPQI to form mercapturic acid [19].

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In present study, among all the studied plants, P. niruri
and M. emerginata has greatest ability to reduced
oxidative stress by increasing glutathione level and
preventing lipid peroxidation in compared to other
studied plants.
All the studied plants extract contain antioxidants and
hepatoprotective activity through regulatory action on
cellular permeability, stability and suppressing oxidative
stress. A number of scientific reports indicated that
certain flavonoids, triterpenoids and steroids have
protective effect on liver due to its antioxidant
properties [30]. Active compound phyllanthin,
hypophyllanthin and lignansniranthin nirtetralin, and
phyltetralin isolated from Phyllanthus niruri leaves
might play key role in hepatoprotective activity. [31]
Phytochemically, Maytenus spp. contains triterpenes,
flavonoids, sesquiterpene and sesquiterpene
evoninoate alkaloids might play role in
hepatoprotective activity. [32]
From our results, it can be concluded that decrease
levels of GSH, catalase activity, and increased serum
marker enzymes and lipid peroxidation level in
paracetamol treated rats was due to hepatocellular
damage. Extract of P. niruri, M. emarginata, E.
alba, A. Vera, S. indicum and A. marmelos afforded
protection from such paracetamol induced liver
damage, where the aqueous extract of Phyllanthus
niruri and Maytenus emarginata has shown the
most pronounced hepatoprotective effect. These
results showed their protective activity in the order
of M. emarginata > P. niruri > A. marmelos > S.
indicum > A. Vera> E. alba.

[9].

[10].
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[14].
[15].
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[18].
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[20].
[21].
[22].

[23].

[24].
[25].
[26].

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