Indo American Journal of Pharmaceutical Research, 2016 ISSN NO: 2231-6876

EVALUATION OF THE HERB-DRUG INTERACTION IN CHICK EMBRYO FOR ANTI-

INFLAMMATORY ACTIVITY
Thorat Rinku D, Joshi Yadhunath M*, Garge Vaibhavi N.
Bharati Vidyapeeth’s College of Pharmacy, Sector-8, C.B.D, Belapur, Navi Mumbai, Maharashtra, India.
ARTICLE INFO ABSTRACT
Article history The present study investigates the interaction between Boerhavia diffusa (Punarnava
Received 24/06/2016 capsules) and Diclofenac sodium powder using chick embryo model for anti-inflammatory
Available online activity. Boerhavia diffusa (Nyctaginaceae) is one of the significant indigenous medicinal
10/07/2016 plant with a broad spectrum of therapeutic activity. The medicinal value of this herb has been
reported for its anti-diabetic and diuretic properties. In different areas of the world, it has also
Keywords been used for analgesic, anti-inflammatory, and carminative activities. The present study was
Boerhavia Diffusa, aimed at evaluation of herb- drug interaction between Boerhavia diffusa and Diclofenac
Punarnava Capsules, sodium for the anti-inflammatory activity using chick embryo model. The inflammation was
Diclofenac Sodium, induced by placing whatman filter paper disc on the chorio-allantoic membrane of an 8-day
Anti-Inflammatory Activity, chick embryo. This model is comparatively cheap, requires little space to perform and only
Chorio-Allantoic Membrane. small quantities of the drugs are required. Concurrently no interaction study has been
performed on this selected herb and drug.

Corresponding author
Joshi Yadhunath M
Professor in Pharmacology,
Bharati Vidyapeeth’s College of Pharmacy,
Sector-8, CBD, Belapur, Navi Mumbai, Maharashtra, India.
yadunath@vsnl.net
27571122, 27572131

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Please cite this article in press as Joshi Yadhunath M et al. Evaluation of The Herb-Drug Interaction in Chick Embryo for Anti-
Inflammatory Activity .Indo American Journal of Pharmaceutical Research.2016:6(06).

Copy right © 2016 This is an Open Access article distributed under the terms of the Indo American journal of Pharmaceutical
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Research, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.

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Vol 6, Issue 06, 2016. Joshi Yadhunath M et al. ISSN NO: 2231-6876

INTRODUCTION
Inflammation is a body defense reaction in order to eliminate or limit the spread of injurious agents which is a local response
to injury of living mammalian tissues. Various components to an inflammatory reaction represent edema formation, leukocyte
infiltration and granuloma formation that can contribute to the associated symptoms and tissue injury. [1]
Boerhavia diffusa is the plant used in present study (Family; Nictaginaceae) is commonly known as punarnava mainly an
herbaceous creeping weed and is widely distributed in the tropical and temperate region of the world. [2] As per Ayurvedic claims it
exhibits a wide range of medicinal properties. The whole plant of B. diffusa has been employed for the treatment of various disorders
like gastrointestinal disorders, liver disorders and heart diseases. In earlier studies of some groups it has shown to have anti-
inflammatory, antimicrobial, immunosuppressive, hepatoprotective, antitumorogenic, antileprotic, antiasthmatic activities and
laxative, diuretic, antiurethritis, anticonvulsant, antifibrinolytic, antinematodal, antibacterial properties. [3- 6]
Diclofenac is a nonsteroidal anti-inflammatory drug (NSAID) of the phenyl acetic acid class with analgesic, anti-
inflammatory, and antipyretic properties. [7]
Experimental studies on the incubated egg, for example the embryo and the chorio-allantoic membrane (CAM), are carried
out frequently. The first system supplying the embryo is the yolk-sac blood vessel system. [8]
The avian chorio-allantoic membrane (CAM) is the outermost extra embryonic membrane lining the non-cellular eggshell
membrane. It is formed by fusion of the splanchnic and the somatic mesoderm of the allantois and the chorion. By day 12 th of
incubation the fused CAM develops and covers the entire surface of the inner shell membrane of the chicken. At day 21 st the chick
will normally hatch. The CAM serves as a support for the extra embryonic respiratory capillaries it actively transports sodium and
chloride from the allantoic sac and calcium from the eggshell into the embryonic vasculature and forms part of the wall of the allantoic
sac that collects excretory products.
For studying biological processes such as tumor transplant experiments, toxicity, more recently angiogenesis, acute and
chronic inflammatory responses the CAM is a common method because of low cost, the simplicity of the surgical procedure, and the
possibility to continuously observe the test site without disrupting it, [9] The chick embryo chorio-allantoic membrane (CAM) was
also used as an in vivo wound healing model. [10]

Figure 1: Candling of egg.

With the help of candling living normal embryos show (figure 1): [11]
 Clearly defined blood vessels with no hemorrhagic areas evident
 Some body movement when stimulated by the candling light
 A generally healthy appearance
 Dead embryo show a ring of blood outlined on the inner surface of the shell.

Fertile eggs of a cross strain were used (Sudanese bantam-English White Leghorn). [12] These were incubated at 36-37°, the
eggs were turned twice daily. In preliminary tests, the initial incubation period varied between 8 and 12 days. The rationale behind
selection of chick embryo CAM model was the advantages such as easy availability, less expensive and no protocol approval with
IAEC over methods involving the use of laboratory animals. One disadvantage is that the slope of the dose-response curve appears to
be inherently shallow and therefore the test may not be suitable for precise biological assay.

MATERIALS AND METHODS COLLECTION OF DRUG AND HERB

The drug Diclofenac sodium was collected from Nutraplus India limited, and herbal formulation of Punarnava (Boerhavia
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diffusa) capsules was collected from Himalaya herbal health care

EXPERIMENTAL ANIMALS
Fertilized Chick Embryo of 2 days old were procured from Central Poultry Development Organization for chick embryo
model. Fertile eggs of a cross strain were used (Sudanese bantam-English White Leghorn). These were incubated at 36-37°; a moist
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atmosphere was maintained by placing dishes of water on alternate shelves in the incubator; the eggs were turned twice daily.

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CHEMICALS AND BIOMATERIALS

DMSO, Diclofenac sodium, Boerhavia diffusa, Sterile filter paper disk.

EXPERIMENTAL DESIGN
Chick embryo chorio-allantoic membrane model
The inflammatory reaction was induced by placing whatman filter paper disc on the chorio-allantoic membrane of an 8-day
chick embryo followed by re-incubation in situ for 4 days. Drug and herb are dissolved in DMSO.
Treatment groups are as followed:
Group I: induced saline (0.1ml)
Group II: disease control induced paper disc with saline (0.1 ml).
Group III: induced paper disc with drug sodium diclofenac (1mg).
Group IV: paper disc with herb Boerhavia-diffusa (1mg). [13]
Group V: interaction group paper disc with drug Diclofenac (1mg) and Boerhavia-diffusa (1mg).

DISC
Whatman No. 1 filter paper was used as Filter paper discs. Any disc with rough or uneven edges was discarded; the discs
were washed in distilled water to remove fragments and then sterilized in Universal screw-topped bottles, approximately 50
discs/bottle, by autoclaving (15 lb/sq. in./20 min). Four smaller discs, each approximately 5 mm diameter, were implanted in each egg.

TECHNIQUE
1. The technique used for dropping the chorio-allantoic membrane was a modification of that described by Beveridge & Burnet
(1946). [14]
2. The egg was mounted on a stand, with the drilled area of shell uppermost; a straight Hagedorn's needle was gently inserted under
one corner of the smaller triangle of shell and this triangle was raised and removed, care being taken to avoid puncturing the shell
membrane during this procedure.
3. A drop of sterile saline was placed on the exposed shell membrane and a small slit made in this membrane (and not through the
closely adjacent chorio-allantoic membrane) by gentle downward pressure with the Hagedorn's needle.
4. By means of a rubber teat, suction was applied to the hole drilled over the air sac and the chorio-allantoic membrane fell away
from the shell membrane, drawing in the drop of saline (Figs. 2 b). The shell and shell membrane circumscribed by the larger
triangle were then removed, and the sterile filter paper disc inserted and carefully lowered on to the exposed membrane (Figs. 2
c).
5. The opening in the shell was sealed with Sellotape and the hole over the air sac region sealed with molten paraffin wax (Fig. 2 d).
The eggs were then reincubated at 370 for 4-6 days, without turning.

Fig: a
Fig: b

Fig: c
Fig: d

Figure 2: (a) Chick embryo showing sacs and membrane; (b) Air sac evacuated and chorio-allantoic membrane dropped; (c)
Filter paper disc placed on the chorio-allantoic membrane; (d) Tri-angular opening through shell and shell membrane sealed
with sellotape.
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6. At the end of this period, the chorio-allantoic membrane was exposed by cutting around the long circumference of the egg
with a pair of curved scissors; the chorio-allantoic membrane remained in the top half of the shell, together with the filter
paper disc. The membrane was gently eased out of the shell using forceps.
7. The filter paper disc and the underlying portion of thickened membrane were dissected out and the disc, together with the
underlying granulation tissue, was placed on a plastic spotting tile and dried overnight at. The dry disc plus granulation tissue
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was subsequently weighed.

8. Specific effects of the drug and herb on the growth of embryos were also observed.

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RESULT AND DISSCUSSION

The result obtained from present study indicate that all the treatment groups showed a dose related reduction in the amount of
granulation tissue growing on the undersurface of the implanted disc. Diclofenac sodium was the most potent of the drugs studied; it
caused significant inhibition of granulation tissue as compared to combination treatment group. Growths of chick embryo were also
affected. It has also been observed that growth of diclofenac treated embryos was normal (fig.5). The disintegrated embryos were
observed in the group treated with Punarnava extract (fig.6). This may be due to the effect of herb on the embryonic development. The
treatment group, with both Punarnava extract and Diclofenac sodium showed slow development of embryos (fig.7). In order to assess
the actual amount of granulation tissue present, the weight of the dried filter paper disc was subtracted from the total weight of the
dried disc plus granulation tissue.

Figure 3: Vehicle Control.

Figure 4: Disease Control.

Figure 5: Diclofenac sodium.

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Vol 6, Issue 06, 2016. Joshi Yadhunath M et al. ISSN NO: 2231-6876

Figure 6: Boerhavia diffusa

Figure 7: Diclofenac sodium + Boerhavia diffusa

CONCLUSION
The presence of a filter paper disc on the chorio-allantoic membrane of an 8-day chick-embryo, when incubated at 370 for 4
days, produced an inflammatory reaction on the membrane. This inflammatory response was significantly reduced when diclofenac
sodium 1mg, Boerhavia diffusa 1mg and combination group was impregnated into the disc prior to implantation. Specific effects of
the drug and herb on the embryos were observed. The test, being economical of materials and laboratory space, is suitable for the
routine screening of potential anti-inflammatory compounds at fixed dose levels.

ACKNOWLEDGEMENT
I wish to express my utmost gratitude and heartfelt appreciation to my guide, Dr. Yadhunath M. Joshi, Associate Professor,
Bharati Vidyapeeth’s College of Pharmacy, for his guidance, invaluable advice, encouragement and full confidence throughout my
project. I am most grateful for all the scientific knowledge I have gained from him and all the fruitful discussions we have had.
I would like to thank Ms. Vaibhavi Garge, my co guide for guiding me throughout my study and Mrs. Swati R. Dhande for her timely
help and support. I specially convey my gratitude to my senior Sejal and my guide-mate Nikhitha Narayanan and Kalpana Waikul.

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