Antibacterial
and Wound Healing Activity of the Leaves of
Annona squamosa
Linn. (Annonaceae)
Chitra
Shenoy*1, M B Patil1 and Ravi Kumar2
1Department of Pharmacognosy and Phytochemistry,
K.L.E.S’s
2Department of Pharmaceutics, K.L.E.S’s
ABSTRACT
Natural remedies from medicinal plants are considered to be effective
and safe alternative treatment for wounds. In traditional literature, it is
found that Annona squamosa leaves
were used as folk medicine for the treatment of wound in different parts of the
world. The leaves of Annona squamosa
(Linn.) were exhaustively
extracted by soxhlet apparatus with different
solvents like petroleum ether, solvent ether, chloroform, alcohol and
chloroform water in ascending order of the polarity. All the five extracts were
subjected to antibacterial screening by using the cup plate method. The
petroleum ether, alcoholic and chloroform water extract showed maximum zone of
inhibition. So these extracts were taken for wound healing activity. The
petroleum ether extracts of Annona squamosa
at a dose of 300 mg/kg b.w. (orally) in all models showed significant results. The
percentage contraction of wound are
at 18 th
day, was found to be (91.12±0.62,
P < 0.05) in excision wound model, wound breaking strength (299.83 ±
5.446 grams, P < 0.05) in incision wound model, granuloma
breaking strength (378.56 ± 5.520, P < 0.05) in granuloma
studies. All the results were significant for different parameters in wound
healing activity when compared with control group. Presence of sterols, flavonoids, tannins in various extracts was also confirmed
by preliminary phytochemical investigation, TLC and
HPTLC methods.
Keywords: Annona squamosa, wound healing activity, antibacterial
activity, petroleum ether extract.
INTRODUCTION
Wounds are inescapable events of life; wound
may arise due to physical, chemical or microbial agents and wound healing has
been one of the earliest medical problems. Healing is essentially a survival
mechanism and represents an attempt to maintain normal anatomical structure and
function. Healing of wound takes place in a direction away from its normal
course and it is common to have none, under or over healing. Treatment is
therefore aimed at either shortening the time required for healing or
minimizing the undesired consequences. Advances in surgical skill and technique
have overcome the latter to some extent.
Wound is a breach in the
normal tissue continuum, resulting in a variety of cellular and molecular sequelae. The basic principles of optimal wound healing
which include minimizing tissue damage, debriding
nonviable tissue, maximizing tissue perfusion and oxygenation, proper nutrition
and moist wound healing environment have been recognized for many years1.
A number of drugs ranging from simple non-expensive analgesics to complex and
expensive chemotherapeutic agents administered in the management of wound
affect healing either positively or negatively2.Aspirin, indomethacin, cytotoxic agents
and immunosuppressant have been proved experimentally to affect healing
negatively3-6.
Management of wound healing, particularly the
under healing is complicated and expensive programme. Research on wound healing
drugs is a developing area in modern biomedical sciences. Several drugs from
plant are known to have wound healing properties. Some of these plants have
been screened scientifically for evaluation of their wound healing activity in
different pharmacological models and patients, but the potential of most
remains unexplored. In a few cases, active chemical constituents were
identified. Hence, there is a dearth of safe, economic and effective prohealing agents for the wound management programme, which
can enhance healing as well as control infection.
The plant Annona
squamosa (annonaceae)
is commonly called as custard apple in English sharifa
in Hindi7. This plant is reputed to possess varied medicinal
properties8. It is used as an insecticidal agent has been
investigated by several workers9. Free radical scavenging activity
of Annona squamosa10 was
reported in the leaf extracts. Hypoglycemic and antidiabetic effect of Annona
squamosa11-12
was reported in the leaf extract. From the bark of Annona
squamosa, a bioactive acetogenin
with anticancer activity13-15 have been isolated. Flavonoids from leaves16, Aporphine
alkaloids17-18, glycoside19 and squamoline20
were isolated from this plant.
In the Ayurvedic system of medicine, herbal extracts but not
purified compounds have been used from centuries because many constituents with
more than one mechanism of action are considered to be beneficial. However no
scientific reports were available on the literature for its wound healing
activity. Thus, the present study is designed to fill up the lacunae in the
literature for its wound healing activity with a view to provide scientific
evidence on modern lines.
In this communication
we report the preliminary phytochemical
investigations of the ethanolic, petroleum ether,
solvent ether, chloroform and chloroform water extract of Annona squamosa leaves, the acute toxicity
studies and their wound healing activity.
MATERIALS AND METHODS
Plant material and Preparation of Extracts
The fresh leaves
of Annona squamosa were
collected in the month of July 2008 from the Belgaum and authenticated by Dr. Harsha Hegde, Chief Botanist,
Indian Council of Medical Research (RMRC), Belgaum branch, shade dried and
powdered then passed from 40# mesh size.
Preparation of Various Extracts of Annona squamosa Linn
Around 1 kg fresh shade dried leaves were powdered and
extracted by hot percolation method by soxhlet
apparatus with five liters of each petroleum ether, solvent ether,
chloroform, alcohol, chloroform water and. The percolation process was
continued until the extraction process was completed (indicated by fade coloured menstrum). All the
extracts finally reduced to dryness at 40oC by Rotovapour
(
PRELIMINARY PHYTOCHEMICAL
INVESTIGATION
All the extracts were screened for the presence of various secondary
metabolites like steroids, alkaloids, carbohydrates, proteins, flavonoids, tannins and glycosides using the standard
methods.
Powder Analysis
The powdered crude drug was subjected to determination
of pH, extractive value, total ash, water soluble ash, acid insoluble ash and
loss on drying as per Indian Pharmacopoeia.
Microorganisms
The test microorganisms
used for the antimicrobial activity screening were 4 bacteria (2 Gram positive) – Bacillus subtilis, Staphylococcus aureus,
(2 Gram negative)-Pseudomonas aeruginosa,
Escherichia coli. These organisms were identified and procured from
National Chemical Laboratory (NCL),
Antibacterial Activity
The agar diffusion method21 was used to
evaluate the antibacterial activity. Bacteria were cultured overnight at 37
o C in Mueller Hinton 10 µl Broth (MHB, Oxoid)
and used as inoculum. A final inoculums, using 100 µl
of suspension containing 108 CFV/ml of bacteria spread on Mueller
Hinton Agar (MHA) medium.
The disc (6 mm in diameter) was impregnated with 10 µl
of 200µl/ml, 150µl/ml, 125 µl/ml, 100 µl/ml, 75 µl/ml and 50 µl/ml of each
extracts and for each organism placed on seeded agar. Streptomycin (200µl/ml,
150µl/ml,125 µl/ml, 100 µl/ml,75 µl/ml and 50 µl/ml) was used as positive
control bacteria. The test plates were incubated at 37 o C for 24h
for bacteria depending on the incubation time required for a visible growth.
Pharmacological activity
Experimental
animals
Albino wistar rats of either sex (150-200g weight) and Swiss
albino mice of either sex (18-22g
weight) were procured from Venkateshwara Enterprises,
Acute
Oral Toxicity study
Swiss albino mice
of either sex (18-22g weight) and of 90 days were used to determine the LD50
of various test extracts. The animals were fasted over night prior to the acute
experimental procedure. The acute oral toxicity study was carried out as per
the guidelines set by Organization for Economic Co-operation and Development
(OECD)22. The LD 50 was found to be more than 3000 mg/ kg
b.w. p.o. in acute toxicity
testing. The therapeutic dose 300mg/ kg b.w. p.o. (ED50) was calculated as 1/10 th of the lethal dose for the purpose of wound
healing investigation.
WOUND HEALING ACTIVITY
Albino wistar rats of either sex (150-200g weight) were selected,
and divided into six groups of six each ( n = 6). Animals were depilated
at the desired site before wounding. They are housed individually with free access
to food and water, the basal food intake and body weights to the nearest gram
were noted. The animals were starved for 12 h prior to wounding. Under light
ether anesthesia wounding was performed semi-aseptically. The first, third and
fifth group served as control similarly second, fourth and sixth groups
received alcoholic, petroleum ether and chloroform water extract by oral route
at a dose of 300 mg/kg body weight by oral route daily for 10 consecutive days
in incision and dead space wound model and for 20 days in the excision wound
model. The suspensions of desired concentrations were prepared in gum acacia 2%
solution, and were administered as per the ED50.
Wound models:
1.
Resutered incision
2.
Excision
wound
3.
Dead
space wound (granuloma studies)
Resutered, excision and dead space wound models in
albino rats, were selected for assessing the wound healing activity. These
parameters were selected because of the easy availability of albino rats and
the simplicity in handling them. The rats were anaesthetized prior to incision
and other surgical procedures, and were
sacrified by exposing to a higher dose of anaesthetic ether prior to determination of the tensile
strengths of the resutured wounds, and the removal of
granuloma tissue (grass pith).
Excision wound
An excision wound was inflicted by cutting away 500 mm2 full
thickness of a pre-determined area on
the depilated back of the rat. Epithelialization
period was noted as the number of days after wounding required for the eschar to fall off leaving no raw wound behind. Wound
contraction rate was monitored by planimetric
measurement of the wound area on alternate days. This was achieved by tracing
the wound on a graph paper on 4
th, 8 th,
12 th,16 th and18 th post wound
days and thereafter daily until healing was complete. Reduction in the
wound area was expressed as percentage of the original wound size23.
Incision wound
On the depilated backs of the animals, two paravertebral
incisions of 6 cm length were made cutting through the full thickness of the
skin. Interrupted sutures, 1 cm apart, were placed to approximate the cut edges
of the skin24. The sutures were removed on the 8th post
wound day and skin breaking strength was measured on the 10th day by
continuous water flow technique of Lee25.
Dead
Space wounds (Granuloma studies)
Physical changes in the granuloma
tissue were studied in this model. Under light ether anaesthesia,
subcutaneous dead space wounds were inflicted in the region of the axilla and grion, by making a
pouch through a small neck in the skin. Granuloma
formation was induced by implanting grass pith and cotton pellet. Cylindrical
grass piths measuring 2.5 cm in length and 0.3 cm diameter were introduced into
the pouch similarly. Each animal received 2 grass piths in different locations.
The wounds were sutured and mopped with an
alcoholic swab. Animals were placed into their individual cages after recovery
from anaesthesia. Excision of the granulation tissue
from the surrounding tissue was performed on the 10th post wounding
day under light ether anaesthetic condition. Granuloma surrounding the grass piths was excised and slit
open by longitudinal rectangular strips. The tensile strength of piece
measuring about 15 mm in length and 8 mm in width (obtained after trimming the
granulation tissue) was determined on 10th post wounding day
continuous constant water flow technique of Lee. The granulation tissue so
harvested was subjected to hydroxyproline estimation.
Their weights were expressed as mg/100 gms body
weight as suggested by Dispaquale and Meli26.
Histopathological studies
A section of the granuloma
tissue was subjected to histopathological examination
to determine the pattern of lay-down for collagen using two special stains i.e.Van Gieson and Masson Trichrome.
STATISTICAL
ANALYSIS
All the results were analyzed by One-way Analysis of Variance (ANOVA) followed by Dunnett’s test. The level of significance was set at
P<0.05.
RESULTS
AND DISCUSSION
The average
percentage yield of various extracts of Annona squamosa is
shown in table 1.
Table
1: The percentage yield of various extracts of Annona squamosa L.
|
Sl. No. |
Extracts |
Nature of extract |
Colour |
Yield (% w/w) |
|
1. |
Petroleum ether |
Semisolid |
Dark yellow |
8.05 |
|
2. |
Solvent ether |
Semisolid |
Dark brown |
5.00 |
|
3. |
Chloroform |
Semisolid |
Dark brown |
7.00 |
|
4. |
Alcohol |
Semisolid |
Dark brown |
17.45 |
|
5. |
Chloroform water |
Semisolid |
Dark yellow |
12.00 |
Powder analysis parameters like pH,
extractive value, total ash, water soluble ash, acid insoluble ash and loss on
drying were determined on the powder of Annona squamosa. In powder analysis ash values are useful in
determining the quality and purity of crude drug, especially in the powder form
and the extractive values are useful for their evaluation, especially when the
constituents of a drug can not be readily estimated by any other means. Further
these values indicate the nature of the constituents present in a crude drug.
The results of physicochemical characterization of Annona squamosa
are presented in table 2.
Table
2: Physicochemical Characterization of Annona squamosa
|
Sl. No. |
Parameters |
Result |
|
1. |
Total ash (%) |
2.25 |
|
2. |
Acid insoluble ash (%) |
1.0 |
|
3. |
Water soluble ash (%) |
1.8 |
|
4. |
Loss on drying (%) |
1.25 |
|
5. |
Extractive value (%) |
9.5 |
|
6. |
pH |
6.9 |
The phytochemical tests revealed that the leaves of the plant
possess Sterols in petroleum ether and solvent ether extract. The other
constituents like flavonoids, tannins, glycosides,
carbohydrates and proteins were found in ethanolic
and chloroform water extract. Leaves of the plant possess alkaloids in
chloroform extracts. The results of phytochemical
screening are given in table3.
Table
3: Phytochemical Screening of different extracts of Annona squamosa
|
Extracts |
Steroids |
Alkaloids |
Glycosides |
Saponin |
Flavonoid |
Tannin |
Carbohydrates |
|
Petroleum Ether |
+++ |
- |
- |
- |
- |
- |
- |
|
Solvent Ether |
++ |
- |
- |
- |
- |
- |
- |
|
Chloroform |
+ |
++ |
- |
- |
- |
- |
- |
|
Alcohol |
- |
+ |
+ |
- |
++ |
++ |
+ |
|
Chloroform water |
- |
- |
+ |
+ |
+ |
+ |
++ |
+++:
high concentration; ++: medium concentration; +: low concentration; - : constituents not detectable
Table4: Results of Acute oral toxicity studies of
various extracts of Annona
squamosa
|
Sl. No |
Extracts |
LD50 (mg/kg) |
ED50(mg/kg) |
|
1. |
Petroleum ether |
3000 |
300 |
|
2. |
Solvent ether |
3000 |
300 |
|
3. |
Chloroform |
3000 |
300 |
|
4. |
Alcohol |
3000 |
300 |
|
5. |
Chloroform water |
3000 |
300 |
The LD50
was found to be more than 3000 mg/ kg b.w. p.o. in acute toxicity testing. The therapeutic dose 300mg/
kg b.w. p.o. was calculated
as 1/10 th of the lethal dose for the
purpose of wound healing investigation. The results of oral toxicity studies of
various extracts of Annona squamosa
is presented in table 4.
Antibacterial
activity was done for all the five, pet ether, solvent ether, chloroform,
ethanol and chloroform water extracts. During antibacterial study chloroform
water, petroleum ether and alcoholic extracts showed maximum zone of inhibition
against almost all organisms in cup plate method. So the chloroform, alcohol
and petroleum ether extract were taken for wound healing activity. The results
of antibacterial activity of various extracts of Annona
squamosa are shown in table 5.
The results of
the excision wound model are given in table 6. In an excision wound model,
petroleum ether extract at a dose 300mg/kg BW p.o. of
Annona squamosa showed significant wound healing activity (% wound contraction on 18th
day (91.12 ± 0.620, P <
0.05) compared to control (39.56± 0.47). It also showed complete epithelization (16.00 ± 0.43 days P < 0.05) when
compared to control (24.66 ± 0.211). The petroleum ether extract showed a scar
area of (9.83 ± 0.479 mm 2, P< 0.05) as compared to control 15.5
± 1.52 mm 2.
The results of incision wound model are given in table 7. In incision study, the petroleum ether
extract showed significant (299.83±5.446, P< 0.05) breaking strength when
compared to control (144.2±8.052).
The results of dead space wound model are given in
table 8. The tensile strengths of the granuloma
tissue were determined by the water flow technique of Lee. Petroleum ether
extract showed highly significant increase in breaking strength (378.56±5.520, P<0.05) when compared to control (157.035±8.765) and petroleum ether also showed significant
increase in the dry weight of granulation tissue (53.88±0.46, P<0.05) as compared to
control (30.42±0.47). Petroleum
ether also showed significant increase in the hydroxyproline
content
(2514.17±21.11, P<0.05) as compared to
control (1395±9.66).
Histological studies of granulation tissue of
the petroleum ether extract treated animals showed significant increase in
collagen deposition with macrophages, tissue edema
and more fibroblasts. (fig.1) than alcohol extract compared to control.
DISCUSSION
Granulation, collagen maturation and scar
formation are some of the many phases of wound healing which run concurrently,
but independent of each other. The use of single model is inadequate and no
reference standard exists that can collectively represent the various phases of
wound healing. Hence three different models have been chosen in our study to
assess the effect of Annona squamosa on wound healing. The wound breaking strength is determined by the rate
of collagen synthesis and more so by the maturation process where there is
covalent binding of collagen fibrils through inter and intra molecular cross
linking. In our study dead space wound model showed significant increase in
breaking strength and hydroxyproline concentration,
and also dry weight of the granulation tissue was significantly increased in Annona squamosa treated group. By
this we can assume that the Annona squamosa might have increased the collagen and also probably have altered the
maturation process, by affecting the cross linking of collagen or improving the
quality of collagen fibrils. The increase in weight in Annona squamosa treated group could
be due to high protein concentration and collagen bundle formation27.
Measurement of the hydroxyproline
could be used as an index for collagen turnover. In the present study
significant increase in the hydroxyproline content of
the granulation of the animals treated with petroleum ether extract and
chloroform extract were observed indicating rapid collagen turnover. Increase
in breaking strength of granulation tissue indicated the enhanced collagen
maturation by increased cross linking. In addition, increase in dry granulation
tissue weight indicated the presence of higher protein content.
The preliminary phytochemical
analysis of leaf extract of Annona squamosa revealed the presence
of flavonoids, saponins28, alkaloids29,
tannins and sterols. Flavonoids are known to reduce
lipid peroxidation not only by preventing or slowing
the onset of cell necrosis but also by improving vascularity.
Hence, any drug that inhibits lipid peroxidation is believed to increase
the viability of collagen fibrils by increasing the strength
of collagen fibres, increasing the circulation, preventing the cell damage and by promoting the DNA synthesis30.
TABLE
5: Results antibacterial activity of various extracts of Annona squamosa
|
Sl. No. |
Name of the Extract |
Zone of Inhibition in mm at
conc. of 200 mg/ 0.1 ml |
|||
|
Bacillus subtilis |
Staphylococcus
aureus |
Pseudomonas aeruginosa |
Escherichia
coli |
||
|
1. |
Petroleum
ether |
20 |
19 |
20 |
22 |
|
2. |
Chloroform |
17 |
16 |
19 |
20 |
|
3. |
Chloroform
water |
19 |
19 |
19 |
21 |
|
4. |
Solvent
ether |
12 |
14 |
14 |
17 |
|
5. |
Alcohol |
20 |
18 |
18 |
20 |
|
6. |
Control
(DMF) |
R |
R |
R |
8 |
|
7 |
Standard |
21 |
20 |
22 |
24 |
Diameter of cup – 6mm; Standard drug –
Streptomycin; DMF - Dimethyl formamide;
R – Resistance
|
Group (N) |
Dose (oral)
|
Excision Wound |
||||||
% Wound Contraction
|
||||||||
|
4th
day |
8th
day |
12th day |
16th
day |
18th day |
Mean size of scar area mm2 |
Period of epithelization (days) |
||
|
Control |
1 ml of 2% Gum acacia |
13.90± 0.32 |
20.04± 0.14 |
23.52± 0.57 |
32.55± 0.06 |
39.56± 0.47 |
15.5± 1.52 |
24.66± 0.211 |
|
Petroleum ether |
300 mg/kg suspended in 2% acacia |
25.19± 0.36 |
30.21± 0.02 |
50.63± 0.35 |
74.36± 1.07 |
91.12± 0.62* |
9.83± 0.479* |
16.00± 0.43* |
|
Chloroform water |
300 mg/kg suspended in 2% acacia |
45.98 ±1.10 |
52.75± 1.23 |
70.46±0.45 |
76.35± 1.39 |
90.21± 0.66 |
10.66± 0.3346 |
19.00± 0.366 |
|
Alcohol |
300 mg/kg suspended in 2% acacia |
39.36± 1.286 |
49.63± 1.300 |
67.47± 0.46 |
72.12± 1.47 |
88.21± 0.67* |
10.11± 0.35* |
18.33± 0.211* |
* indicates significant difference at P<0.05 when
compared to control. Values are Mean ± SEM from 6 animals in each group), Data
analyzed by One-way ANOVA followed Dunnett’s test.
Table7: Influence of various extracts of Annona squamosa on
healing of Incision wound
|
Group (n) |
Dose (oral) |
Wound breaking strength (g) |
|
Control |
1 ml of 2% Gum acacia |
144.2±8.052 |
|
Petroleum ether |
300 mg/kg suspended in 2% acacia |
299.83±5.446* |
|
Chloroform water |
300 mg/kg suspended in 2% acacia |
280.67±4.213 |
|
Alcohol |
300 mg/kg suspended in 2% acacia |
263.63±4.48* |
* indicates significant difference at P<0.05 when
compared to control. Values are Mean ± SEM from 6 animals in each group), Data
analyzed by One-way ANOVA followed Dunnett’s test.
Table 8: influence of various
extracts of Annona Squamosa on
healing of dead space wound
|
Group (n) |
Dose (oral) |
Breaking
strength (g) |
Granulation
tissue dry weight (mg/100g) |
Hydroxyproline
(µg/100mg) |
|
Control |
1 ml of 2% Gum acacia |
157.035± 8.765 |
30.42±0.47 |
1395±9.66 |
|
Petroleum ether |
300 mg/kg suspended in 2% acacia |
378.56± 5.520* |
53.88±0.46* |
2514.17±21.11* |
|
Chloroform water |
300 mg/kg suspended in 2% acacia |
289.8± 4.351 |
30.98±0.85 |
1465±11.76 |
|
Alcohol |
300 mg/kg suspended in 2% acacia |
283.82± 4.44* |
40.99±0.37 |
1910±5.63* |
* indicates significant difference at P<0.05 when
compared to control. Values are Mean ± SEM from 6 animals in each group), Data
analyzed by One-way ANOVA followed Dunnett’s test.
Flavonoids31-32, triterpenoids33
are also known to promote the wound-healing
process mainly due to their astringent and antimicrobial property, which seems to be responsible for wound contraction and
increased rate of epithelialisation. Tannins the main components of many plant
extracts, act as free radical scavengers34-39. Research into the
role of antioxidants from plant extracts in wound healing has been published
widely40.
CONCLUSION
The use of Annona
squamosa in
Indian traditional systems of medicine for various skin diseases, has been
justified by this work, as it showed a wound healing potential and commendable
activity against several microorganisms. These findings could justify, at least
partially, the inclusion of this plant in the management of wound healing in
folk medicine. Since the role of free radicals and antioxidants in wound
healing are very clearly defined, wound healing potential Annona squamosa may be partly due to the potent antioxidant activity of the plant.
Further experiments are needed to test the effect of this plant in the
treatment of chronic wounds. To conclude, the petroleum ether extract of Annona squamosa exhibited significant wound healing activity in excision,
incision, burn and dead space wound model. Hence the present findings provide
scientific evidence that petroleum ether extract of Annona
squamosa as a potent wound
healer.
Figure1.
Histology of the Granulation tissue of Pet ether treated animal showing
more collagen and less macrophages (Vangeison stain).
ACKNOWLEDGMENT
The authors are thankful to Dr. F.V. Manvi,
Principal, K.L.E.S’s
REFERENCES
1. Pierce GF, Mustoe
TA. Pharmacologic enhancement of wound healing.
Annu Rev Med.1995; 46: 467–481.
2. Prasad D, Rao CM.
Wound healing profile of ketorolac, metronidazole and
tinidazole administered postsurgically.
3. Lee KH. Studies on the mechanism of action of
salicylates III. Effect of vitamin A on the wound
healing retardation action of aspirin. J
Pharma Sci.
1968; 57: 1238–1240.
4. Rao CM, Ramesh KV, Bairy KL, Kulkarni DR. A simple
method to quantify maturation of wound collagen.
5. Raju S, Kulkarni DR.
Vitamin A reverses the wound healing suppressant effect of cyclophosphamide.
6. Holla RK, Sequeira RP, Kulkarni DR. Cyclosporin and
wound healing.
7.
Morton J. Sugar apple. Fruits Warm Climate.1987:
69-72.
8.
Watt G.Periodical Experts:
A Dictionary of the Economic Products of
9.
Cheema PS, Dixit RS, Koshi T, Perti SL. Insecticidal
properties of the seed oil of Annona squamosa Linn. J
10. Shirwaikar A, Rajendran K, Kumar CD. In vitro antioxidant studies of Annona squamosa Linn.
leaves. Indian J Exp Biol. 2004; 42: 803-7.
11. Kaleem M, Asif M, Ahmed QU, Bano B. Antidiabetic and antioxidant activity of Annona squamosa extract
in streptozotocin-induced diabetic rats.
12. Gupta RK, Kesari AN, Murthy PS, Chandra R, Tandon
V, Watal G. Hypoglycemic and antidiabetic
effect of ethanolic extract of leaves of Annona squamosa L.
in experimental animals. J Ethnopharmacol. 2005;
99:75-81.
13.
14.
15. Li XH, Hui YH, Rupprecht JK, Liu YM, Wood KV, Smith DL, Chang CJ, McLaughlin
JL. Bullatacin, bullatacinone,
and squamone, a new bioactive acetogenin,
from the bark of Annona squamosa.
J Nat Prod. 1990; 53:81-6.
16. Seetharaman TR. Flavonoids from the leaves of Annona
squamosa and Polyalthia
longifolia. Fitoterapia.
1986; 57:189-198.
17. Bhakuni DS, Tewari S, Dhar MM. Aporphine alkaloids of Annona
squamosa. Phytochemistry. 1972;11:1819–1822.
18.
19.
Forgacs P, Desconclois JF, Provost R, Tiberghienet
Touche A. Un Nouvel Heteroside Nitre Extrait D’ Annonasquamosa. Phytochemistry. 1980 ;
19:1251–1252.
20. Yang TH, Chi-Ming
C. Structure of squamolone, a novel diazepine from Annona squamosa L. J Chin Chem Soc (
21.
22.
Organization for Economic Co-operation and
Development, revised draft guidelines 423,“OECD Guidelines for the testing of
chemicals” Revised document-October 2000.
23.
Werner S, Breededen M, Hubner G, Greenhalgh D G and Longaker M T. Introduction of keratinocyte
growth factor expression is reduced and delayed during wound healing in the
genetically diabetic mouse. J Investig Dermatol. 1994; 103:469.
24.
Ehrlich HP, Hunt TK. The effect of cortisone and
anabolic steroids on the tensile strength of healing wounds. Ann Surg.
1968;167:324.
25.
Lee KH. Studies on the mechanism of action of salicylate retardation of wound healing by aspirin. J Pharma Sci. 1968;57:1042-3.
26. Dispasquale G, Meli A. Effect of body weight changes on the formation of
cotton pellet induced granuloma. J Pharm Pharmacol. 1965;17:379-82.
27. Annie Shirweiker, Radhika Shenoy, Udupa AL, Udupa SL, Somashekhar Shetty. Wound healing
property of leaves of Hyptis suaveolens
with supportive role of antioxidant enzymes. Indian J Exp Biol. 2003;
41: 238–241.
28. Gulcin I, Mshvidadze
V, Gepdiremen
A and Elias R. Antioxidant activity of saponins isolated from Ivy; alpha-heredin,
hederasaponin –C, hederacolchiside
–E and hederacolchiside –F. Plant Medica.
2004;70:6, 551.
29. Marjorie M.C. plant products as antimicrobial
agents. Clinic microbiology reviews, 1999; 12: 54.
30. Getie M, Gebre Mariam T, Reitz R, Neubert RH. Evaluation of the release profiles of flavonoids from topical formulations of the crude extract
of the leaves of Dodonea viscosa (Sapindaceae). Pharmazie. 2002;57:320-322.
31. Tsuchiya H, Sato M, Miyazaki T, Fujiwara S, Tanigaki S, Ohyama M, Tanaka T, Iinuma M. Comparative
study on the antibacterial activity
of phytochemical flavanones
against methicillin resistant Staphylococcus aureus. J Ethnopharmacol. 1996:50:27-34.
32. .Suchiya H, Sato M,
Miyazaki S, Fujwara S and Linum
M. comparative study of the antibacterial activity of phytochemical
flavanones against methicillin
resistant Staphylococcus aureus, J Ethanopharmacl.
1996; 50:27.
33. Scortichini M,
34. Bekerecioglu M, Tercan M, Ozyazan I. The effect
of Ginkgo biloba (Egb
761) as a free radical scavenger on the survival of skin flaps in rats. Scand J
Plast Reconstr Hand Surg.
1998; 32:135–139.
35. Marja P. Kahkonen, Anu I. Hopia, Heikki J. Vuorela, Jussi-Pekka Rauha, Kalevi Pihlaja
Tytti S. Kujala Marina Heinonen. Antioxidant Activity of Plant Extracts Containing
Phenolic Compounds, J Agric Food Chem. 1999; 47:
3954- 3962.
36. Raquel P, Laura B, Fulgencio
SC. Antioxidant
Activity of Dietary Polyphenols As Determined by a
Modified Ferric Reducing/Antioxidant Power Assay. J Agric Food Chem. 2000;
48:3396-3402.
37. Andrea G, Mirella N, Alessandro B and Cristina S. Antioxidant
Activity of Different Phenolic Fractions Separated
from an Italian Red Wine, J. Agric. Food Chem 1998;
46: 2.
38. Tran VH, Hughes
MA, Cherry GW. The effects of polyphenolic extract
from Cudrania cochinchinenesis
on cell response to oxidative damage caused by H2O2 and xanthine
oxidase. Abstract 27, First Joint Meeting of Chinese
and European Tissue Repair Society, September 22–27,
39. Dutta NK, Shastry MS. Pharmacological action of A. bracteolata Retz. on the uterus.
40. Hwang JK, Kong TW, Baek
NI, Pyun YR. Alphaglycosidase inhibitory activity of hexagalloyl glucose from the
galls of Quercus infectoria.
Planta Med. 2000; 66: 273–274.
Received on 03.05.2009
Accepted on 22.05.2009
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Research Journal of Pharmacognosy and Phytochemistry. 1(1): July.-Aug. 2009, 44-50