Research J. Pharm. and Tech.

11(2): February 2018

ISSN 0974-3618 (Print) www.rjptonline.org

0974-360X (Online)

RESEARCH ARTICLE

Comparative Hylauronidase enzyme activity of Ayurvedic formulation

Triphala guggulu
Alok Sharma1*, Dinesh Kumar Mishra2, Karunakar Shukla3
1
Research scholar, Uttarakhand Technical University, Dehradun, Uttarakhand
1
Mahakal Institute of Pharmaceutical Studies, Ujjain (M.P.)
2
Associate Professor, School of Pharmacy and Technology Management, SVKM’s NMIMS, Shirpur (Maharastra)
3
Principal, Royal Institute of Management and Advanced Studies, Ratlam (M.P.)
*Corresponding Author E-mail: aalokpharma@yahoo.com, dineshkumar.mishra@nmims.edu,
drshukla19@gmail.com,

ABSTRACT:
A herbal formulation Triphala Guggulu was prepared based on the method described in Ayurvedic Formulary of
India. It contains This triphala guggulu includes Amla (Emblica officinalis), Bibhitaki (Terminalia Bellerica),
Haritaki (Terminalia Chebula), Pippali (piper longum), Guggulu (Commiphora mukul) plants. Maximum plant
was used in preparation of ayurvedic formulation shows anti inflammatory activities and formulation also use for
treatment of inflammation and in arthritis.The aqueous extract of TG exhibited a maximum hyaluronidase
enzyme inhibition of 83.90% and marketed formulations TG-1,TG-2 and TG-3 exhibit maximum maximum
hyaluronidase enzyme inhibition of 83.86%,83.87% and83.89% respectively. The results of this study suggest
that the Hyaluronidase enzyme inhibition of Triphala Guggulu may explain its use in joints related problems like
Rheumatoid arthritis, Gout and in inflammation of joints.

KEYWORDS: Hyaluronidase, Triphala Guggulu

INTRODUCTION: Triphala Guggulu is an Ayurvedic formulation

The term “Ayurveda” combines the Sanskrit words ayur
(life) and veda (science or knowledge) which means
“The science of life”. Ayurveda was the first holistic
system of diagnosis and treatment integrating nutrition,
hygiene, rejuvenation and herbal medicine. The most of
the Ayurvedic formulation are lacking in their defined
quality control parameters and method of its evaluation.
The World Health Organization (WHO) has emphasized
the need to ensure the quality of medicinal plant
products by using modern controlled technique and
applying suitable standards. The use of Ayurvedic
formulations has led to the sudden increase in the
number of Ayurvedic drug manufactures due to the
toxicity and side effcts of allopathic medicines.
Received on 18.09.2017 Modified on 02.11.2017
Accepted on 24.12.2017 © RJPT All right reserved
Research J. Pharm. &Tech 2018; 11(2):463-465.
DOI: 10.5958/0974-360X.2018.00085.9
containing Guggulu (Commiphora mukul) is the major
ingredient along with Triphala ingredients1,2,3.
The medicinal oleoresins produced by Commiphora
mukul were known as myrrh. In Ayurvedic literature,
myrrh oleoresins are referred to as guggulu. Guggulu
destroys bodily wastes and toxins, boosts metabolism
and strength, and rejuvenates the body. Excessive bodily
waste and toxins plays a major role in the aetiopathology
of rheumatoid arthritis. A study reported the anti-
inflamatory activity of crystalline steroids from C.
mukul resin4,5. Guggulu is an exudates obtained from the
plant Commiphora mukul. Preparations having the
exudates as main effective ingredient are known as
Guggulu. There are five different varieties of Guggulu
described in the Ayurvedic texts. However two of the
varieties, namely, Mahisaksha and Kanaka Guggulu are
usually preferred for medicinal preparations.
Mahisaksha Guggulu is dark greenish brown and
Kanaka Guggulu is yellowish brown in color6.

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 Research J. Pharm. and Tech. 11(2): February 2018

Triphala guggulu is a polyherbal ayurvedic Triphala Guggulu Preparation:

formulation.This medicines is guggulu kalp and contain
guggulu more than other ingredients use to make this
medicines. As the name itself suggests that medicine
contains triphala and guggulu .triphala improves
digestion, cure constipations and detoxifies bodies.
Guggulu gives relief in inflammation and pain.Triphala
guggulu is indicated in low metabolism, obesity, piles,
fistula, inflammation and gout etc7, 8, 9.
The formulation taken for the present study is as
compiled in Sarangdhara samhita This triphala guggulu
includes Amla (Emblica officinalis), Bibhitaki
(Terminalia Bellerica), Haritaki (Terminalia Chebula),
Pippali (piper longum), Guggulu (Commiphora mukul)
plants. Phenolic compounds of methanolic extract of
Amla (Emblica officinalis) may serve as a potential
herbal drug for amelioration of acute inflammation due
to their modulatory action on free radicals10, 11. Haritaki
(Terminalia Chebula) used in the treatment of arthritic
disorders and suggest that radical quenching may be one
of the mechanisms for its anti-inflammatory activity
and also show antioxidant activity12,13,14. Pippali (piper
longum) dried fruit's oil was taken for the study. The
results obtained indicate that the Piper longum oil had
significant antiinflammatory activity in rats15,16,17.
Guggulu (Commiphora mukul) has been extensively
studied in inflammation and pain in bones, joints,
muscles and related connective tissues18-22.
The Components of Triphala were also evaluated for
their inhibitory activities on hyaluronidase and
collagenase. This is the fi rst report to show that the T2
component of Triphala (T. chebula) is a highly potent
hyaluronidase and collagenase inhibitor23.
MATERIAL METHODS:
Assay was developed to investigation this hypothesis.
First, we tested the effects of the Triphala guggulu home
made formulation (TG) and its marketed formulations
TG-1, TG-2, TG-3) on the activity of hyaluronidase, an
enzyme that destroys the hyaluronic acid (HA) backbone
of cartilage matrix. Indeed, the serum level of HA is a
reliable biomarker of Rheumatoid arthritis progression24.
Table-1 Composition of Triphala Guggulu (Ayurvedic formulary of India)
S.No. Hindi name Botanical name
1 Haritaki Terminalia chebula
2 Bibhitaka Terminalia belerica
3 amlaki Emblica officinalis
4 Krishna pipali piper longum
5 Guggulu Commiphora mukul
Table-2 Percent inhibition of hyaluronidase by TG formulations
Aqueous extracts
Test sample Without TG With TG
Hyaluronic acid (HA) 0.489± 0.033 0.538± 0.040
HA + enzyme 0.057± 0.031 0.514± 0.039
Enzyme activity 88.34% 4.44%
% enzyme inhibition 83.9% (88.34–4.44)
Triphala guggulu (TG), expressed as mean ± SD of 3 experiments
Wash, dry and powder the ingredients Amla (Emblica
officinalis), Bibhitaki (Terminalia Bellerica), Haritaki
(Terminalia Chebula), Pippali (piper longum), of the
formulation composition separately and pass through
sieve number 85, weigh them separately in the required
quantities and mix. Crush weighed quantity of Guggulu-
suddha, add fine powder of other mixed ingredients to it
and pound well and mix properly. Add Ghrita to smooth
the progress of the pounding and carry on pounding till a
semi-solid consistently mixed mass of suitable plasticity
is obtained. for 8 to 10 h. This Roll the Vatis on flat
surface to round them by circular motion of palm
covered with a glove and smeared with Ghrita or use
suitable mechanical device (Table 1). Dry the rounded
Vat is in a tray dryer at not more than a temperature
600ºC. formulation is known as TG. A sample of this
formulation was used for this study. Three marketed
preparation of Triphala Guggulu also use for
comparative study of Triphala Guggulu. This
formulation was known as TG-1, TG-2 and TG-3. A
sample of this marketed formulation also used for study.
Preparation of Aqueous extract:
Aqueous extracts: Triphala guggulu TG were solubilized
in distilled water (20–50 mg/ml) by limited autoclaving
(5 lbpressure, 8 min) to yield extracts. Same method was
also used for marketed preparation TG-1,TG-2 and TG-
3 respectively.
Hyaluronidase Assay:
Hyaluronidase was assayed by a highly sensitive
spectrophotometric method, based on precipitation of
HA with cetylpyridinium chloride, which is used for
high throughput screening for hyaluronidase inhibitors 25.
Enzyme (800 U/ml) and HA substrate (0.40 mg/ml)
were incubated at 37°C for 1 h. Enzyme activity was
measured by monitoring the percentage of undigested
HA substrate in the cetylpyridinium chloride precipitate
at absorbance 415nm after the enzyme reaction (Table
2).
Family Part used Quantity
Combretaceae pericarp of mature fruits 45 g
Combretaceae pericarp of dried ripe fruits 45g
Euphorbiaceae pericarp of dried mature fruits 45g
piperaceae Fruit 45g
brusaraceae Dried exudate 240g
With TG1 With TG2 With TG3
0.535± 0.020 0.536± 0.018 0.539± 0.016
0.511± 0.032 0.512± 0.032 0.515± 0.031
4.48% 4.47% 4.45%
83.86% (88.34–4.48) 83.87% (88.34–4.47) 83.89% (88.34–4.45)
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 Research J. Pharm. and Tech. 11(2): February 2018
Formulae were used for calculations:
% Enzyme activity =
(100%) – {A415 nm of HA + enzyme/A415 nm of HA x 100}
RESULT AND DISCUSSION:
Triphala guggulu contain Triphala and Guggulu.
Triphala synergized with the guggulu resin in enhanced
the hyaluronidase activities of the TG, TG-1, TG-2 and
TG-3 formulations.
Percentage inhibition of hyaluronidase enzyme for
Triphala guggulu formulation found to be for TG
83.90% and for TG-1, TG-2 and TG-3 are 83.86%,
83.87% and 83.89% respectively. Maximum
hyaluronidase enzyme inhibition found in TG and
minimum hyaluronidase enzyme inhibition found in TG-
3. In all formulation hyaluronidase enzyme inhibition
found to be close to 83%.
This triphala guggulu includes Amla (Emblica
officinalis), Bibhitaki (Terminalia Bellerica), Haritaki
(Terminalia Chebula), Pippali (piper longum), Guggulu
(Commiphora mukul) plants. Phenolic compounds of
methanolic extract of Amla (Emblica officinalis) it serve
as a potential herbal drug for acute inflammation due to
their modulatory action on free radicals. Haritaki
(Terminalia Chebula) used in the treatment of arthritic
disorders Piper longum oil had significant
antiinflammatory activity in rats. Guggulu (Commiphora
mukul) has been extensively studied in inflammation and
pain in bones, joints, muscles and related connective
tissues.
This result become important when we used costly
injections of hyaluronic acid into the synovial cavity is
administered to patients whose suffer from chronic
arthritis. It may become a alternative.
CONCLUSION:
Percentage inhibition of hyaluronidase enzyme study on
Triphala Gugglu In house and marketed formulations are
studied. The result shows that triphala guggulu may be
use in certain type of diseases like joints pain,
inflammation, Rheumatoid Arthritis. Threfore, it has
been concluded that the formulation prepared by
traditional method reported in Ayurvedic
Pharmacopoeia is the best formulation in respect of that
activity.
REFERENCES:
1. Ayurvedic Pharmacoepia of India, first edition, ministry of health
and family welfare, new delhi, ebook, 2009.
2. Jain Tripti, and Dashora Kamlesh, Development of fingerprinting
method for Amlakyadi Churna: Spectrophotometric approach,
Research Journal of Pharmacognosy and Phytochemistry. 4(2):
March -April 2012, 61-63.
3. Swapna E. etal, Evaluation of Antioxidant activity of Marketed
Ayurvedic Formulations Balarishta, Research Journal of
Pharmacognosy and Phytochemistry. 7(2): April-June, 2015, 107-
110.
465
4. Arora R B, Kapoor V, Gupta S K and Sharma R C, Isolation of a
crystalline steroidal compound from Commiphora mukul and its
anti-inflammatory activity; Indian J. Exp. Biol.1971, 9, 403–404.
5. Christian Praneeta, Effectiveness of Guggul Application on
Intravenous Infiltration; Int. J. Nur. Edu. and Research 4(1): Jan.-
Mar., 2016; Page 73-78.
6. Ayurvedic Pharmacoepia of India, part II, first edition, ministry of
health and family welfare, New Delhi,ebook, 2008,I,136.
7. Ayurvedic Pharmacoepia of India, part I, second edition, ministry
of health and family welfare, New Delhi, ebook,2003, 203.
8. Jain Vishal etal, TLC Densitometric Methods for Quantification of
Gallic Acid in Triphala Churna for Routine Quality Control;
Research J. Pharm. and Tech. 4(2): February 2011; Page 230-233.
9. Mahajan Anil D. and Pai Nandini R., Simultaneous Determination
of Eight Phytoconstituents in Triphala churna by HPLC–DAD;
Research Journal of Pharmacognosy and Phytochemistry. 3(2):
MarchApril 2011, 62-66.
10. Middha S.K. et al, Toxicological Evaluation of Emblica officinalis
Fruit Extract and its Anti-inflammatory and Free Radical
Scavenging Properties; Pharmacogn Mag. 2015 Oct;11(Suppl
3):S427-33.
11. Bothiraja C etal, In Vitro Anti-HIV-Type 1 and Antioxidant
activity of Emblica officinalis; Research J. Pharm. and Tech.2 (3):
July-Sept. 2009,;Page 556-558.
12. Anwesa Bag et al, Anti-inflammatory, anti-lipid peroxidative,
antioxidant and membrane stabilizing activities of hydroalcoholic
extract of Terminalia chebula fruits; Pharm Biol, 2013; 51(12):
1515–1520.
13. Meena A.K. etal, Physicochemical and Preliminary Phytochemical
Studies on the Fruit of Terminalia chebula Retz.; Asian J.
Research Chem. 3(4): Oct. - Dec. 2010; Page 844-846.
14. Vaghela J.S. and Sisodia S.S., In Vitro Antioxidant Activity of
Terminalia chebula Fruit Extracts; Research J. Pharm. and Tech.
4(12): Dec. 2011; Page 1835-1843.
15. A. Kumar et al, Antiinflammatory Activity of Piper longum Fruit
Oil; Indian J Pharm Sci. 2009 Jul-Aug; 71(4): 454–456.
16. Kumari M. et al, 2012, Anti-inflammatory activity of two varieties
of Pippali (Piper longum Linn.); Ayu. 2012 Apr;33(2):307-10.
doi: 10.4103/0974-8520.105258.
17. Verma Prabha and sutarkar Suresh kumar, Dyslipidemic and
Antioxidant activity of Piper longum extract in hyperlipemic rats;
Research J. Pharm. and Tech. 8(12): Dec., 2015; Page 1609-1614.
18. Duwiejua M etal, Anti-inflammatory activity of resins from some
species of the plant family Burseraceae; Planta Med, 59 (1) 1993
12.
19. Sharma J N and Sharma J N, Comparison of the anti-
inflammatory activity of Commiphora mukul (an indigenous drug)
with those of phenylbutazone and ibuprofen in experimental
arthritis induced by mycobacterial adjuvant, Arzneimittel
forschung, (1977) 27(7) 1455.
20. Kimura I etal, New triterpenes, myrrhanol A and myrrhanone A,
from guggul-gum resins, and their potent anti-inflammatory effect
on adjuvant-induced air-pouch granuloma of mice; Bio org Med
Chem Lett, (2001) 11(8) 985.
21. Singh B B, etal, The effectiveness of Commiphora mukul for
osteoarthritis of the knee: an outcomes study; Altern Ther Hlth
Med, (2003) 9(3) 74.
22. Panda Purnendu etal, Anti-Inflammatory Activity of Singhanada
guggulu and Vatari guggulu: A Pharmacological Study; Research
J. Pharmacology and Pharmacodynamics; 3(1): 2011; 17-18
23. Venil N sumantran et al. Hyaluronidase and collagenase inhibitory
activities of the herbal formulation Triphala guggulu; J. Biosci.,
June 2007, 32(4), 755–761.
24. Pavelka K, Forejtova S and Olejarova M 2004 Hyaluronic acid
levels may have predictive value for the progression of knee
osteoarthritis; Osteoarthritis Cartilage 2004 ,21, 277–278.
25. Tung J S, Mark G E and Hollis G F A microplate assay for
hyaluronidase and hyaluronidase inhibitors; Anal. Biochem. 1994,
223,149–152.
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