EVALUATION OF IN VITRO ANTI INFLAMMATORY ACTIVITY OF TRAYODASHANG GUGGULU – AN AYURVEDIC FORMULATION IN COMPARISION WITH ALLOPATHIC DRUGS

Abstract

The objective was to assess the in vitro anti inflammatory activity of the ayurvedic formulation Trayodashang Guggulu in comparison to commercially available allopathic drugs. Anti inflammatory activity was estimated by inhibition of protein denaturation method, which in turn was proceeded by adapting two different types of proteins which are egg albumin and bovine serum albumin. These were incubated along with actives and incubated in carefully controlled experimental conditions. The ayurvedic formulation that is prepared prior was compared with six commercially available allopathic standards. The study results showed inhibition of protein according to the concentration of the active used. The ayurvedic formulation showed prominent anti inflammatory activity better than the allopathic drugs at all concentrations that are selected between 200 to 1000µg/ml except when compared to Diclofenac. This study showed that the ayurvedic combination formulation showed good anti inflammatory properties when tested for extent of protein denaturation. But the effect due to each of its constituents should be evaluated further.

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EVALUATION OF IN VITRO ANTI-INFLAMMATORY ACTIVITY OF TRAYODASHANG GUGGULU:
AN AYURVEDIC FORMULATION IN COMPARISON WITH ALLOPATHIC DRUGS
M Sahiti 1, BM Gurupadayya 2* and TK Dinesh 3
1PG Scholar, Department of Pharmaceutical Chemistry, JSS College of Pharmacy, JSS Academy of Higher Education
& Research, Mysuru, India
2Professor, Department of Pharmaceutical Chemistry, JSS College of Pharmacy, JSS Academy of Higher Education &
Research, Mysuru, India
3Medical Officer, Government Ayurvedic Hi-tech Panchakarma Hospital, Mysuru, India
Received on: 02/04/19 Accepted on: 29/05/19
*Corresponding author
E-mail: bmgurupadayya@jssuni.edu.in
DOI: 10.7897/2277-4343.100370
ABSTRACT
The main objective was to assess the in vitro anti inflammatory activity of the Ayurvedic formulation Trayodashang Guggulu in comparison to
commercially available allopathic drugs. Anti inflammatory activity was estimated by inhibition of protein denaturation method, which in turn was
preceded by adapting two different types of proteins which are egg albumin and bovine serum albumin. These were incubated along with actives and
incubated in carefully controlled experimental conditions. The Ayurvedic formulation that is prepared prior was compared with six commercially
available allopathic standards. The study results showed inhibition of protein according to the concentration of the active used. The Ayurvedic
formulation showed prominent anti inflammatory activity better than the allopathic drugs at all concentrations that are selected between 200 to
1000µg/ml except when compared to Diclofenac. This study showed that the Ayurvedic combination formulation showed good anti inflammatory
properties when tested for extent of protein denaturation. But the effect due to each of its constituents should be evaluated further.
Keywords: Anti inflammatory, Trayodashang Guggulu, Allopathic drugs, Protein denaturation.
INTRODUCTION
The groups of drugs that are specifically for the treatment which
reduces inflammatory reactions are called anti-inflammatory
drugs. Half of the analgesics are anti-inflammatory drugs, which
act by reducing swelling, unlike the opioids which act on central
nervous system by blocking the pain signaling mechanism of
brain. Nonsteroidal anti-inflammatory drugs (NSAIDs) attenuate
soreness by impeding the cyclooxygenase (COX) enzyme. Since,
prostaglandins are synthesized from COX enzymes, which in turn
cause inflammation. In whole, the NSAIDs prevent the
prostaglandins from ever being synthesized, reducing or
eliminating the pain. A few commonly used NSAIDs are Aspirin,
Ibuprofen, and Diclofenac etc1. The newer selective COX
inhibitors are not categorized along with the older, traditional
NSAIDS. The newer specific COX-inhibitors are not classified
together with the traditional NSAIDs despite the fact that they
presumptively have the similar mechanism of action2.
On the contrary, there are some NSAIDS, which have good
analgesic properties, but have very minimal anti inflammatory
effect. For instance, paracetamol has a greater pain reducing
properties than inflammation properties. Inflammation, pain
erythema is caused as a result of human body’s natural defense
mechanism against injuries. To relieve from these effects,
NSAIDS play a effective role by various inflammatory pathways.
But at the same time they can cause a broad category of side
effects which are very much undesirable such as gastric
ulceration, stroke, myocardial infarction and sometimes toxicity
at varied levels3.
On the other hand, most of the herbal formulations or the herbal
drugs possess similar effectiveness while treating both acute as
well as chronic inflammation and pain syndromes with almost
none or very low side effects. So to say because of the above
mentioned reasons and due to consideration of the side effect
profiles of the allopathic anti inflammatory drugs these days,
there has been a greater interest in usage of herbal remedies and
natural compounds4. In fact, many of the currently preferred
natural compounds work by the same mechanism by inhibiting
the inflammatory pathway such as NSAIDS5.
In this work we established the fact of how much better are the
commonly used allopathic anti inflammatory drugs to a herbal
Ayurvedic formulation which is potentially known to be a good
anti inflammatory remedy by a in-vitro anti inflammatory test.
This is to prove the efficacy difference between the herbal and
allopathic formulations.
Trayodashang Guggulu (also spelled as Trayodashang Guggulu)
is Guggulu based herbal formulation. It aids in strengthening of
ligaments, muscles, bones and joints. It is efficient in problems
related to the musculoskeletal and nervous system. Along with
Gokshurad Guggulu and Ashwagandha powder in combination,
it is known to have greater effect against osteoarthritis. It is
advantageous in all kinds of pain such as gout, paralysis, sciatic
pain, hemiplegia, joint pain, etc. it is proved to be working well
when pain and weakness are associated side by side.
Therapeutic Indications
Trayodashang Guggulu is helpful in following health conditions.
• Brain & Nerves: Paralysis, Facial paralysis, Hemiplegia,
Sciatica.
• Pain of neurological origin: Muscle, Bones & Joints,
Osteoarthritis

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• Bone pain: Osteoporosis or low bone mineral density
• Muscle spasm: Backache
Trayodashang Guggulu Benefits & Uses
The primary action of Trayodashang Guggulu is on joints, bones,
nerves and muscles. For this reason it plays a major role in
treatment of all the above stated conditions. Here are some
primary benefits and medicinal applications of Trayodashang
Guggulu.
• Paralytic disorders (facial paralysis or hemiplegia)
According to an ancient Ayurvedic literature, the main
indication of Trayodashang Guggulu is its use in paralytic
disorders. It works well when you start the medication in the
first month of paralytic attack. Sometimes it helps even in the
chronic cases, but then its outcome is low.
• Osteoarthritis and Osteoporosis (low bone mineral density)
It helps in cessation of wear and tear of joints and cartilages
in case of osteoarthritis. However, in case of osteoporosis
Trayodashang Guggulu do not increase bone mineral density
directly, so we require supplements of calcium, zinc,
magnesium and vitamin D. It prevents from fractures and
bone injuries by giving strength to bones.
Trayodashang Guggulu Dosage
The nominal dose of Trayodashang Guggulu starts from 750mg
and not more than 1.5grams twice a day. Although a dose less
than this is not much effective in osteoarthritis and paralytic
disorders, however, a combination of mineral supplements and
Lakshadi Guggulu can be used as supportive therapy. Loss of
appetite, Belching, Stomach upset and Indigestion is a few side
effects that are observed only with high dosage that is more than
6 grams per day6.
Table 1: Plant Material- Trayodashang Guggulu Ingredients
Ayurvedic
medicine
Botanical name
Quantity
Abha
Acacia nilotica Linn. / Indica
/ Arabica Willd
1 part
Ashwagandha
Withania somnifera
1 part
Hapusha
Juniperus communis Linn.
1 part
Guduchi
Tinospora cordifolia
1 part
Shatavari
Asparagus racemosus
1 part
Gokshura
Tribulus terrestris
1 part
Vriddhadaru
Argyreia speciosa
1 part
Rasna
Pluchea lanceolata
1 part
Shatahva
Anethum sowa Kurz
1 part
Shati
Hedychium spicatum
1 part
Yamani
Trachyspermum ammi
1 part
Shunthi
Zingiber officinale
1 part
Guggulu
Commiphora mukul
12 parts
Ghee
Clarified butter
1 part
Fine powder of all these medicinal ingredients is added to
Guggulu, compounded and made into tablets.
MATERIALS AND METHODS
Apparatus
For the evaluation of in vitro anti inflammatory activity Antech
GT sonicator was used for the solubilization of the allopathic and
the herbal formulation. An Incubator of Labline technologies was
used for the incubation of the protein samples which is done
before heating in electric heated water bath. A U.V spectrometer
of Shimadzu, UV1700 was used for checking the extent of
denaturation of protein in the heated samples.
Drugs and chemicals
All pure drugs Diclofenac, Aspirin, Paracetamol, Ibuprofen,
Lornxicam, Piroxicam were procured from Sigma Aldrich, India
and other chemicals and reagents are purchased from SD Fine
Chemicals, Mumbai of analytical grade.
Preparation of Guggulu Shodhana
Common impurities such as stone, bark, glass, etc. are withdrawn
and then it is broken into small pieces. After that, the cleaned and
broken pieces tied in a small cloth and are boiled in Dola-Yantra
which contains Triphala decoction. The boiling of this mixture is
done until Guggulu has turned into a soft mass. Later, it was
retrieved onto a cloth and was spread onto a smooth wooden
board which was previously smeared with ghee. It was dried in a
dust free place and stored in an environment free from moisture
and heat until it is further used for Trayodashang Guggulu
preparation.
Method of Preparation of tablets
The above mentioned herbal drugs (table1) are dried and made
into fine powders separately. These drugs and purified Guggulu
put into the iron mortar and mixed well until a homogenous
mixture formed. While mixing above mentioned; ghee also added
for smoothening of the mixture. After the formation of mixture
pills were made. Criterion to determine the final stage before
making pills is that it should not stick to the fingers when rolled.
The pills were dried in shade. These pills were kept in air tight
containers. These pills were not exposed to sunlight.
EVALUATION OF ANTI INFLAMMATORY ACTIVITY
BY IN-VITRO METHOD
Inhibition of protein denaturation method
The reaction mixture (5mL) contains 3mL of 1% egg albumin
solution in phosphate buffered saline (PBS, pH 6.5) and 2mL of
varying concentrations (200µg/ml and 1000µg/ml) of all the
selected allopathic standard drugs. A Similar volume of double-
distilled water served as control. Then the mixtures were
incubated at (37±2ºC) in a BOD incubated for 30 min and then
heated at 60º C for 10 min. After cooling, their absorbance was
measured at 660 nm by using the vehicle as blank. The treatment
is similar for determination of absorbance for herbal formulation.
The percentage inhibition of protein denaturation was calculated
by using the following formula:
% Inhibition of protein denaturation = Absorbance of control –
Absorbance of sample / Absorbance of control X 100
Where, Vt = absorbance of the test sample, Vc = absorbance of
control.
The percentage inhibition of herbal drug is estimated and
compared with standard drugs by solubilizing an entire tablet (in
10ml distilled water) and further it was sonicated for 30 minutes
and filtered with Whatman filter paper. From the filtered solution
a 2ml aliquot is added to the reaction mixture. Similarly, the same
procedure is used for Bovine serum albumin to compare both the
types of proteins using the same method (inhibition of protein
denaturation method)
RESULTS AND DISCUSSION
In the present research, the anti inflammatory effect is evaluated
against denaturation of egg albumin and bovine serum albumin.
% Inhibition = 100 x (Vt /Vc - 1)

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The results are compiled in table 4 and table 5. However, prior
to establishing the comparative study of the allopathic drugs with
the herbal formulation, Diclofenac sodium was analyzed whether
the drugs have concentration dependent inhibition of protein
denaturation or not (with both egg albumin and bovine serum
albumin). The results of this primary analysis are shown in table
2, 3 and figure 1, 2.
Table 2: Relationship between concentration of Drug and % Inhibition of Protein Denaturation (Egg Albumin Method)
S. No
Concentration
Absorbance
% Inhibition
1
200 µg/ml
0.310
27.05
2
400 µg/ml
0.273
35.76
3
600 µg/ml
0.252
40.70
4
800 µg/ml
0.211
50.35
5
1000 µg/ml
0.170
60.01
6
Control
0.425
-
Table 3: Relationship between concentration of Drug and % Inhibition of Protein Denaturation (Bovine Serum Albumin Method)
S. No
Concentration
Absorbance
% Inhibition
1
200 µg/ml
0.030
46.42
2
400 µg/ml
0.026
53.57
3
600 µg/ml
0.023
58.92
4
800 µg/ml
0.019
66.07
5
1000 µg/ml
0.016
71.42
6
Control
0.056
Figure 1: Linearity relationship between concentration of drug and
%inhibition of protein (egg albumin) denaturation. X-axis:
concentration (µg/ml), Y-axis: %inhibition of protein denaturation
Figure 2: Linearity relationship between concentration of drug and
%inhibition of protein (bovine serum albumin) denaturation. X-
axis: concentration (µg/ml), Y- axis: % inhibition of protein
denaturation.
Table 4: Results of % Inhibition with Egg Albumin Method
S. No
Drug name
Absorbance of
Conc. (200µg/ml)
Absorbance of
Conc. (1000µg/ml)
% Inhibition
(200µg/ml)
% Inhibition
(1000µg/ml)
1
Diclofenac sodium
0.308
0.17
27.18
59.81
2
Aspirin
0.334
0.296
26.6
42.9
3
Piroxicam
0.361
0.323
17.17
30.95
4
Ibuprofen
0.372
0.334
13.7
26.6
5
Lornoxicam
0.371
0.333
14
27
6
Paracetamol
0.406
0.368
4.18
14.94
7
Trayodashang
Guggulu
1 full tablet = 0.283
49.46%
8
Control
0.423
Table 5: Results of % Inhibition with Bovine Serum Albumin Method
S. No
Drug name
Absorbance of
conc. (200µg/ml)
Absorbance of
conc.(1000µg/ml)
% inhibition
with (200µg/ml)
% inhibition
with (1000µg/ml)
1
Diclofenac sodium
0.028
0.017
50.00
69.64
2
Aspirin
0.039
0.019
30.3
66.07
3
Piroxicam
0.04
0.02
28.57
64.28
4
Ibuprofen
0.034
0.023
39.28
58.92
5
Lornaxicam
0.047
0.037
16.07
33.92
6
Paracetamol
0.046
0.040
17.8
28.57
7
Trayodashang
Guggulu
1 full tablet = 0.018
67.85%
8
Control
0.056
Always there are complications while using animal subjects for
in vivo pharmacological research, like ethical matters and lack of
rationale for the use of animals when there are some other
methods that can also be used and investigated for the desired

M"Sahiti"et"al"/"Int."J."Res."Ayurveda"Pharm."10"(3),"2019"
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results. Hence forth in the current study a bioassay by protein
denaturation method was adopted as a part of in vitro estimation
of anti-inflammatory property of the formulated Ayurvedic drug7.
For inflammatory and arthritic conditions, denaturation of tissue
proteins is one of the well proved causes. Hence it is known that
auto antigen production in arthritic cases is due to protein
denaturation8, 9.
That is why the chemical entities that have the ability to
counteract against denaturation process can be potentially used as
anti inflammatory drugs10. In table 2 and 3, the trend was
observed that as we increased the concentration of the drug
against control showed the absorbance slumped which represents
the stabilization of proteins in the presence of anti inflammatory
drug. So as a result the % inhibition of protein denaturation hiked
up as we increased the concentration of the drug (Diclofenac
sodium). From the table 4 and 5 it was clear that Diclofenac was
more active than the other anti inflammatory counterparts having
at least 50% inhibition even at the lowest concentration of
200µg/ml. These results were supported by conducting the same
procedure on two different types of proteins, i.e., egg albumin and
bovine serum albumin. The greater the turbidity of the sample,
the greater is the denaturation of the protein that happened due to
heating process11.
Although, the turbidity of the test samples (herbal / allopathic
drug), of all concentrations had been always less than that of
control. This fall in turbidities is because of the decrease in
concentration of herbal/allopathic drug in the reaction mixture,
which resulted in decreased turbidity12. Therefore, from the
results of the present preliminary study, it can be concluded that
herbal drug Trayodashang Guggulu possessed marked in vitro
anti-inflammatory effect against the denaturation of protein.
Further definitive studies are necessary to deduce the mechanisms
and constituents behind its anti-inflammatory actions. When 2ml
of the herbal formulation extract was used in the reaction mixture,
it showed a good % inhibition of protein denaturation than all the
other allopathic drugs except Diclofenac sodium. It remained to
be having the highest efficiency when compared to all the other
drugs at both 200µg/ml and 1000µg/ml. This gives a conclusion
that the Ayurvedic formulation has good, acceptable anti
inflammatory properties.
While comparing the in vitro anti inflammatory activity of the
individual herbal drugs in this formulation from the previously
reported research literature review showed that the individual
drugs have a considerable anti inflammatory activity but the
combination is found to be better.
For instance, consider Ashwagandha from the results of the
studies carried out previously. It was concluded that it possessed
a marked anti inflammatory activity against denaturation of
protein. The effect of Ashwangandha was more when compared
to Diclofenac13. Similarly, when (Tribulus terrestris Linn) was
tested for anti inflammatory activity 500µg/ml concentration of
fruit kashaya showed a marked inhibition of almost 80% which is
less than Diclofenac percentage inhibition14.
For instance, consider Ashwagandha from the results of the
present preliminary study, it can be concluded that it possessed
marked anti-inflammatory effect against the denaturation of
protein in vitro. The present results exhibited a concentration
dependent inhibition of protein (albumin) denaturation by the
Ashwagandha extract. The effect of diclofenac sodium was found
to be less when compared with the test extract. From this analysis,
it can be concluded that the Ayurvedic formulation has effective
anti inflammatory properties even though it is slightly less when
compared to diclofenac. It can be evident that this Ayurvedic
formulation can be used for the inflammatory indications with the
least number of side effects for a longer duration when compared
to the allopathic formulations.
CONCLUSION
This is a preliminary study, we developed in vitro anti
inflammatory activity of the Ayurvedic formulation
Trayodashang Guggulu in comparison to commercially available
allopathic drugs and further investigations are required to find an
active component of the extract and to confirm the mechanism of
action. The effect of concentration of test agent on the viscosity
behavior of denatured protein dispersion requires further studies.
The effect may be due to synergistic effect rather than single
constituent. The present findings corroborated this property of
Trayodashang Guggulu in vitro. Further definitive studies are
necessary to ascertain the mechanisms and constituents behind its
anti-inflammatory actions both in vivo and in vitro.
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Cite this article as:
M Sahiti et al. Evaluation of In vitro anti-inflammatory activity
of Trayodashang guggulu: An Ayurvedic formulation in
comparison with allopathic drugs. Int. J. Res. Ayurveda Pharm.
2019;10(3):92-96 http://dx.doi.org/10.7897/2277-4343.100370
Source of support: Nil, Conflict of interest: None Declared
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