Biochemistry

HYALURONIDASE: 2021’S AGING ENZYME – AN ETHICAL

DISCUSSION - Review
Andrea Monsalve 1, Sofía Bárcenas 2 and Mónica Porcell 3
1
Ingeniería química; andreamoch@unisabana.edu.co
2
Ingeniería química; paulabagi@unisabana.edu.co
3
Ingeniería química; monicapogo@unisabana.edu.co

Abstract
Aging is a natural process that every human being goes through. The decrease in the concentration of hyaluronic acid in
the body over the years is one of the main factors to which the onset of physical effects such as wrinkles is attributed.
This decrease is mainly due to the action of hyaluronidase (HYAL) as an enzyme that breaks down hyaluronic acid
(HA). The use of HA dermal fillers was implemented as a tool to combat or delay this effect; however, this solution is
not permanent because it is well received for human body and as the HA and HYAL are naturally synthetized for the
body. In the present review we evaluate the enzymatic activity of HYAL in the presence and absence of inhibitors with
the objective of determined the best inhibitor until now, as well as the extraction methods, sources of origin and the
ethical implications involved in the extraction, application in industries and inhibition processes. It was determined that
Vitamin C is the best inhibitor found so far, however, the inhibitory part of it is still unknown. It was also determined
that inhibitors analyzed have in common the presence of carbonyl groups or aromatic rings in their structure and that the
highest inhibition percentage is attributed to inhibitors with origin on natural sources.

Key Words: Aging, degradation, ethics, hyaluronidase, hyaluronic acid, inhibition, vitamin C.

Introduction of macromolecules, consisting of proteins,

The skin extracellular matrix (ECM) plays a proteoglycans and glycoproteins that confer the
fundamental role in the protection, nutrition, and structural properties of cells and tissues [2]. Among one
regulation of various cellular mechanisms, activating or of the main components of the extracellular matrix are
deactivating the processes of cell growth, cell death, glycosaminoglycans (GAGs), with hyaluronic acid
adhesion, invasion, gene expression and cell (HA) being the only GAG that is not covalently bound
differentiation [1]. It is composed of a complex network to a protein to form a proteoglycan [3]. It is a high

1
molecular weight non-sulfated polysaccharide
composed of repeating polymeric disaccharides of D-
glucuronic acid and N-acetyl-D-glucosamine linked by
alternating β (1-3) and β (1-4) bonds, present especially
in connective tissues, but also in nervous tissue and
epithelia [4]. It is an extremely hydrophilic molecule,
which gives it a high hydration capacity, contributing to
the viscoelastic properties of the skin, providing tissues
with resistance to mechanical stress and lubrication [5].
50% of HA present in the body is found in the dermis,
ranging from concentrations of 0.5 mg/g to 1 mg/g [6].
Unfortunately, the presence of this polysaccharide in the
body decreases progressively over the years because its
degradation is necessary for the permeabilization of
ECM, generating the decrease of moisture and laxity in
the skin, promoting skin aging [7].
One of the main degradative agents of this acid
are hyaluronidases (HYAL). In humans, six types of
HYALs have been identified: HYAL-1, -2, -3, -4, PH-
20 and NYALP1 [8]. Enzymes capable of releasing the
oligosaccharides present in HA by breaking down
hyaluronic acid into monosaccharides by cleaving at
their glycosidic bonds [9]. This generates multiple
effects on tissues, decreasing the high viscosity and
lubricating quality of hyaluronic acid. To prevent these
effects, cosmetic injection of HA dermal fillers has
emerged, being one of the main strategies used for the
prevention of aging [10]. In addition, these enzymes are
causative of various diseases, it has been shown that
their concentration in the body can be affected by
diseases such as Alzheimer's disease and dementia.
Nevertheless, HYALs can be employed in medical
practice to increase tissue absorption of various drugs,
as they act as a "spreading factor" facilitating the
diffusion of different substances injected
subcutaneously, such as anesthesia and antiviral
vaccines [11, 12].
Despite this innovative tool, multiple findings
have been made to find the right inhibitor for these
enzymes. Since these fillers are not permanent and HA
is degraded by them after 18 months [13]. Currently
several types of inhibitors have been developed from
plant and fruit extracts, however, all of them are
characterized by very low yields, around 37% - 50%
[14]. However, vitamin C has been one of the inhibitors
that has shown a higher yield (60-80%), as well as a
higher inhibitory activity on HYAL, presenting
inhibition percentages of up to 97% [15, 16]. It is
thought that its inhibition is due to its antioxidant
activity, although it is not totally attributed to this cause,
since there are other antioxidants that have not been able
to inhibit it or have inhibited it minimally, such as
quercetin [17].
The present review aims to study the enzymatic
activity of hyaluronidase in the presence and absence of
inhibitors and its capacity to degrade hyaluronic acid.
Additionally, it explains the ways of obtaining
hyaluronidase, its sources of origin and the ethical
implications of the extraction process and its application
in different industries.
Aging and antiaging
Aging is a physiological phenomenon defined
as a progressive failure of metabolic processes [18] but
despite this, it is called a natural process in humans [19].
Aging is caused by different theories, among them the
free radical theory, the neuroendocrine theory, the
telomerase aging theory, the wear and tear theory, the
rate of life theory, the accumulation of waste products
theory, the cross-linking theory, the immunological
theory, the error and repair theory and the order of
disorder theory [20, 21]. The common denominator of
these theories lies in the presence of molecules such as
free radicals, telomeres, and enzymes such as
hyaluronidase, accompanied by external factors such as
exposure to x-rays and UV rays, resulting in premature
aging that is phenotypically evident in wrinkles and skin
blemishes [22].
Hyaluronic acid and hyaluronidase
Hyaluronidase is a natural enzyme, produced by
humans, capable of locally degrading hyaluronic acid,
specifically its β1-4 bond between hyaluronic acid and N-
acetylglucosamine [23]. In the aesthetic
dermatological industry, it is beneficially used in
treatments against incorrect hyaluronic acid filler
injection [24]. Hyaluronic acid (HA) is
glycosaminoglycan (GAG), not bound to a core protein,
naturally synthesized by the human being that fulfills
important functions in the skin such as hydration and
anti-aging effect. HA production in the skin decreases
with age and exposure to solar radiation, affecting the
transport of ions and macromolecules in
extracellular space due to decreased hydration [23].
Almost proportionally to the decrease in HA there is
evidence of an increase in hyaluronidase, which is
involved in aging processes due to the release of
cytokines by keratinocytes which degrade the three-
dimensional network of fibers of the dermis and
phenotypically are evidenced by wrinkles, skin
blemishes and loss of elasticity [25]. Hyaluronidase is
found naturally in the dermis, which is divided into
layers, specifically it is found in the epidermis and is
compartmentalized and especially present in the basal
and spinosum stratum inside the cells and in the spaces
between them. In the dermo-epidermal junction only
hyaluronic acid is found and from there the molecule
migrates to the extracellular space and to the upper
layers of the skin, enriching keratinocytes and in the
dermis the highest amount of hyaluronic acid is found
and at the highest concentration because it is
synthesized specifically in the plasma membrane of the
fibroplasts, very present in this layer of the dermis [26].
In addition to aging, hyaluronidase has also
been found to be present in cancer and non-cancer
related tumor growth [10]. A recent colorectal cancer
investigation showed the presence of different types of
hyaluronidase (Hyal-1, -2, -3 and PH-20) especially in
advanced cancer stages; each type of hyaluronidase was
and present in different tissue distribution which allowed the
identification of different cancer isoforms at certain
stages [27].
a
Hyal-1 is found within different cell types
partially compartmentalized in vesicles that traffic in a
manner different from clathrin or caveolin endosomal
and in the lysosome. The presence of this enzyme has
been attributed to the capacity of metastasis in some
the types of cancer, especially in colorectal, genitourinary,
ovarian, breast, pancreatic and lung cancers; moreover,
it has not been found in its normal structure but its
capacity to generate variants called Hyal1-v1, Hyal1-v3
and Hyal3-v1 has been evidenced [28]. By performing
different studies on Hyal-2 it has been possible to
identify that it becomes more present in premalignant
and malignant melanomas but when the cancer is in an
advanced stage the concentration of this isoenzyme
decreases considerably as the body develops specific
antibodies to Hyal-2, for example the Hyal2ex2-3
variant was associated with early gastric tumor lines and
Hyal-2 was seen present in many stages related to
astrocytoma tumor growth [29, 30, 31]. The Hyal-3, -4
and PH-20 isoenzymes are categorized as the three least
studied members of the hyaluronidase family, of which
it has been determined that Hyal-3 is not anchored to the
plasma membrane of the cell as Hyal-4 and PH-20 are,
and of the latter two only PH-20 shows similar activity
to hyaluronidase, but specifically in the testis and does
not change tumor cell lines. Although the relationship
of these isoenzymes with cancer has not been deeply
studied, their presence has been identified in the phases
of tumor growth and in remote metastasis [28, 32].
3

Hyaluronidase has also been beneficially used
in human ocular surgery as an anesthesia potentiator,
and in cosmetic surgery as a treatment for wrong
hyaluronic acid injection, but more frequently and
safely in cosmetic surgery because surgeon and
esthetician have more experience in this stage plus the
numerous investigations about this topic. Its use in
human ocular surgery has still been investigated so it
can be advantageously used [33, 34, 35]. However, this
enzyme has shown negatively effects in pig vocal cord
involvement causing dehydration and decreasing its
tangent moduli in the kinetic study from Ꜫ=0.3 when the
vocal cord is correctly hydrated to Ꜫ=0.15 when it is
dehydrated because of presence of hyaluronidase [36].
Sleep apnea is another topic that hyaluronidase has
affect suggesting that an increase of Hyal-1 levels and
an accelerated degradation of the High Molecular
Weight Hyaluronic Acid (HMW-HA) caused for the
enzyme or for alterations in the hyaluronan metabolism
increase levels of chronic hypoxia and inflammatory
process generating sleep apnea in patients [37].
Alzheimer and dementia are also related with increase
of hyaluronidase levels in the body; as mentioned before
hyaluronidase is extremely related with aging and high
levels of hyaluronidase can make human body to get
older in least time and allows the development of some
mental diseases and even in renal and vascular risk
factors [11], same thing has happened with collagen
degradation. Collagen can be stored in human dentin
and considering that hyaluronidase can be present in
saliva, a study showed that collagen was degraded for
the action of increased concentrations of hyaluronidase
in saliva and this process was not associated with
presence of any bacteria because it was isolated from
microorganisms that could affect the study [38].
Obtention, quantification and purification
Hyaluronidases have been identified in
eukaryotes and prokaryotes as they can be found in
many of our tissues. For example, in the kidney, spleen,
skin, liver, placenta and testes [25]. Also, in venoms of
snakes, lizards, bees, fish, scorpions or body liquids as
sperm, tear liquid and blood. It can be found in some
bacteria like fungi or invertebrate animals as leech or
crustaceans [39]. The samples vary on molecular
weight, substrate specificity, optimal conditions, and
catalytic mechanism, depending on the source of origin
(Fig.1) [40]. Therefore, these extractions can be
classified in three different groups. First, the
hyaluronate 4-glycanohydrolases are responsible of
degrading hyaluronan by the breach of beta-1,4-
glycosidic bond, with tetra-saccharides as main product
[41]. These enzymes can also depolymerize chondroitin
and dermatan sulfate [42]. They can be found in
lysosomes, testicles, and bee venom. The second type
are the 3-glycanohydrolases, found in the salivary
glands of invertebrate animals as leech or crustaceans.
These enzymes hydrolyze the beta-1,3-glycosidic bond
of sugar fragments in glucuronic acid, producing tetra-
saccharides [43]. The third group are hyaluronate
lyases, commonly known as microbial hyaluronidases.
They produce an unsaturated disaccharide by an
elimination reaction and can be isolated from
microorganisms as Micrococcus, Streptococcus,
Clostridium and Streptomyces [41].
 Fig 1. Classification of hyaluronidases [39]

Based on the amino acid sequence, the glucosamine. They have been studied because
hyaluronidases can also be classified in two main hyaluronate lyases are consider a factor that facilitate
families, from eukaryotes or prokaryotes. spreading of bacteria in host tissues. Human infection
Hyaluronidases from eukaryotes are mainly obtain from by S. agalactiae is one of the major causes of serios
mammals or bovine testicles [44]. Several diseases as meningitis or septicemia [47]. That´s why
hyaluronidase’s samples can be studied inside the the design and development of inhibitors of this enzyme
mammalian category. Human testicular hyaluronidase become important.
can be found because is required for sperm penetration
through oocyte´s cell layer. It is known as PH-20 protein The extraction of hyaluronidase is considered a
or SPAM 1 (sperm adhesion molecule 1). Within these very expensive and difficult process, as the source,
samples, there were two hyaluronidase-like found with cellular tissue, must be alive and fresh [48]. It has been
about 40% identical amino acid sequence, Hyal-1, and proved that hyaluronidase extraction from frozen
Hyal-2. They differ on their function inside the body. testicles or seminal plasma is not recommended nor
Hyal-1 produces oligosaccharides, while Hyal-2 possible, as it inactivates rapidly upon release or during
degrades high molecular weight HA [45]. They have the extraction [49]. One of the extraction methods
been widely studied as Hyal-1, as it is believed they are applied is the Hahn´s method in testicles obtained as
an inactivated suppressor gene in many tobacco-related soon as possible after dead. Also, high pressure
lung tumors. Hyal-2 was found to promote tumor cell homogenization has been studied, but is not
cycling. On the other hand, bovine testicular recommended as it demands thermal, chemical, and
hyaluronidase (BTH) breaks beta-1,4-glycoside bonds other potential disnaturing factors. Centrifugation and
of hyaluronan, acting as endo-glucanohydrolase [46]. precipitation are one of the most common methods.
Usually, homogenizing the source with sodium acetate
The second family are the samples from buffer, NaCl and phenylmethanesulfonylfluoride,
prokaryotes as Streptococcus pneumoniae and S. centrifugated at 26000 g for 75 min recovering the
agalactiae. Both enzymes break beta-1,4-glycosidic supernatant. Then subjected to an affinity
bond between D-glucuronic acid and N-acetyl-D- chromatography column [50]. Quantified with Lowry´s

5
method using bovine serum albumin as standard [51].
The most common purification route consists of
(NH4)2SO4 fractionation separation of insoluble
protein, Column I. Sephadex G-100, Column II. DEAE-
cellulose and Column III. Sephadex G-75 [50].
Inhibition
Due to all the processes in which hyaluronidase
in involved, its inhibition has been extensively studied.
Hyaluronidase inhibitors are described as a potent
regulator capable of maintaining hyaluronic acid
homeostasis, which in turn could serve as anti-
inflammatory, antioxidant, antimicrobial and even
anticancer agents, and provide a deeply insight into the
functioning of hyaluronidase-type enzymes in terms of
hyaluronic acid metabolism [52].
Some of the inhibitors that have been identified
are phlorotannin, alginate and fucoidan extracted from
two types of Sargassum seaweeds and from the Eisenia
arborea thallus, with a maximum anti-hyaluronidase
percentage of 37.67% with the phlorotannin extracted
from a leaf of S. tennerimum, also showing antioxidant
activity with anti-aging potential. The study showed that
increased anti-hyaluronidase activity was associated
with decreased production of N-acetylglucosamine,
concluding that the 3 inhibitors are natural origin
Fig 2. Determined structure of seven acylated iridoid glycosides used as inhibitors [55]
compounds with commercial potential in the medicine
and aesthetic industry [53].
The hydroalcoholic extract of the Piper
Peltatum plant was also evaluated as an inhibitor against
bovine hyaluronidase and extracted from the venom of
two types of snakes (Naja naja atra and B. atrox) by
performing different tests with respect to the
compounds of the extract (flavonoids, phenols, and
alkaloids). All compounds showed an inhibition higher
than 50% with bovine hyaluronidase, between 4.69 and
23.05% with Bothrops atrox venom and between 14.59
and 40.17% for Naja naja atra venom and from these
results it could be concluded that low inhibitor
concentrations result in high inhibition percentages and
that the inhibitory activity is given by the complex of all
the compounds of the extract and not as a singularity
[54].
Another study evaluated the inhibitory activity
of seven acylated iridoid glycosides extracted from
Picrorhiza kurroa rhizomes on hyaluronidase and
determined the chemical structure of each of them (Fig.
2), showing a maximum inhibition of 61.8% by the
Picrorhizaoside E with a concentration of 100µM of
inhibitor [55].
 Vitamin C is an acid synthesized from glucose
and galactose only by plants and some animals. It is
highly recognized for its many beneficial functions in
the human body as an antioxidant used in the treatment
of diseases ranging from the common cold to cancer
[56], for this reason, the inhibition of hyaluronidase with
vitamin C has been a current subject of study. In a
related study, 6-O-acylated vitamin C derivatives have
shown capacity of inhibiting the enzyme but with
selectivity on bacterial lyases [57], however, in another
study the inhibition with vitamin C derivatives of bovine
sperm hyaluronidase was applied and the maximum
inhibition found was 97% with an inhibitor
concentration of 5%, making it to date the best inhibitor
of this enzyme [16].
The inhibition of enzymes related to anti-aging
has been necessary not only because of their
counterproductive effects on human health but also as a
treatment for different diseases, for example, a study
that sought to inhibit enzymes related to anti-aging
processes found that proper inhibition should lower the
levels of captopril, perindopril and enalapril to control
the development of diseases such as Alzheimer’s
disease [11] and as this example there are numerous
studies of inhibitors applied in hyaluronidase that are
shown in table 1. Therefore, enzyme inhibition becomes
a challenge for today’s scientists and researchers. To
have accurate and certain results it is necessary to
understand the kinetics of the enzyme, in this specific
case hyaluronidase, and its substrate, in this case
hyaluronic acid.

Table 1. Comparison of inhibitors and its effect in different origin hyaluronidase

Origin of Chemical Inhibitor
Inhibitor Concentration Reference
hyaluronidase definition Percentage
Bovine testes Phlorotannin from Leaf of Polyphenol 37.67% 22.405 µg/mg [53]
S. tennerimum
Bovine testes Flavonoids from Piper Flavonoid 69.75 – 72.78% 1000-6.25 mg/mL [54]
peltatum hydroalcoholic
extract
Venom from snake Flavonoids from Piper Flavonoid 14.59 – 40.17% 1000-6.25 mg/mL [54]
(Naja naja atra) peltatum hydroalcoholic
extract
Venom from snake Flavonoids from Piper Flavonoid 4.69 – 23.05% 1000-6.25 mg/mL [54]
(Bothrops atrox) peltatum hydroalcoholic
extract
Not specified Picrorhizaoside E from Acylated iridoid 61.8% 100 µM [55]
Picrorhiza kurroa glycoside
Bovine testes Vitamin C Antioxidant 97% 5% [16]
Bovine testes Gallic Acid Antioxidant 42.4% 5 µg/mL [58]
Bovine testes Sodium Alginate Polysaccharide 78% 22.3 µg/mL [59]
Bovine testes Oleanolic Acid Fatty acid 76.5% 219.2 µg/mL [60]
Bovine testes Quercetin Flavonoid 29% Not specified [17]
Snake venom Quercetin Flavonoid 8% Not specified [17]
Bovine testes Tranxanox Anti – allergic drug 85% 0.04 mM [61]
Bovine testes Indole carboxamides Synthetic molecule 2 – 6% 50 µM (pH 7) [62]

Kinetic necessary to analyze the kinetics of each of them to

Considering that there are different determine the similarity or differentiation between each
hyaluronidase origins, as mentioned before, it is hyaluronidase and its subtypes. The most used enzyme

7
in the different industries is bovine’s testicular origin,
which in a study carried out showed to follow the
Michaelis-Menten enzymatic kinetic using hyaluronic
acid as substrate and obtaining Km and Vmax values of
2.23 mM and 19.85 U/mL, respectively [63]. Another
study reached the same kinetic model result, but the
values reported for Km and Vmax were 0.16 mM and
0.05 mM/min (Fig. 3), respectively, in this study the
analytical method used was fluorescence, considered
one of the most effective and sensible methods for
measurement enzymatic activity at present [64].
An inhibition applied to hyaluronidase with
heparin at near physiological conditions showed non-
Fig 3. Michaelis-Menten graph of hyaluronidase activity [64]
The enzymatic activity carried out on the
hyaluronidase obtained from scorpion venom did not
showed a Michaelis-Menten kinetic model and could
not be attributed to any of the known models due to their
structural complexity. However, its inhibition made
with five different types of Brazilian antivenoms
extracted from animal fluids is shown in Fig. 4 and
competitive behavior by Lineweaver-Burke
linearization. Such behavior is attributed to the presence
of syndecans, which are components present in high
concentrations in cell membranes and in pericular
matrix [65]. Another inhibition carried out with an
hydrangenol derivatives called BT-1 showed strong
inhibition activity but with this inhibitor the enzyme no
longer followed the Michaelis-Menten kinetic model,
and the reason could not be determined [66], however,
this allowed to get to the conclusion of that natural
inhibitors showed a higher percentage of inhibition than
inhibitors get from drugs.
showed that when the concentration of the inhibitor was
higher the inhibitory activity tended to decrease [15].
Plus, the enzymatic activity was measurement
spectrophotometrically at 490nm because it was used a
colorimetric method that develop an orange color and
this one can absorb blue color that corresponds to a
wavelength between 450 and 500nm [67].
 Fig 4. Different hyaluronidase scorpion venoms enzymatic activity [48]

At last, but not least, the inhibition made with vitamin C
in testicular bovine hyaluronidase could not be
attributed to any defined kinetic model, but showed
none inhibition at 1mM and a 50% inhibition of the
bacterial type of enzymes [68], this allowed the
conclusion that higher inhibitor concentrations are
counterproductive and significantly reduced the
inhibitory activity that’s the reason why concentration
between 5 and 250 µM are the right ones if a higher
inhibitory percentage is required. However, in other
study of inhibition with different concentrations (6.22
ng/mL, 10.6 ng/mL and 0.2 µg/mL) of ascorbic acid, the
enzymatic activity was defined by the model BTH and
it was useful to define the amino acid sequence
alignment with the sequences of hyaluronidase so the
mechanism of action could be thoroughly understood
[69].

Fig 5. Different concentrations of ascorbic acid (6.22 ng/mL (▲), 10.6 ng/mL (●) and 0.2 µg/mL (■)) as inhibitor and its
influence in the enzymatic activity of hyaluronidase [69].

9
Ethics
As it was discussed earlier in the article,
hyaluronidase inhibition and obtention have a lot of
industrial and medical applications. It can be used for
Alzheimer’s and cancer treatments, antiaging or even
for ocular surgery. However, it´s obtention must be from
specific cellular tissues of animals, bacteria, or humans.
To what extent is it valid to treat diseases knowing it is
cure may involve killing or mistreating animals? It is
true that some hyaluronidase extraction methods do not
involve animals, but studies show these alternatives are
the ones with the lowest yields, from around 18% to
23%. For industrial processes, the extractions are
optimized by using slaughtered animals with a time of
death lower than three hours, by removing bovine
testicles, snake tails, fangs removal or bee´s sting [39,
44]. It is important to know that the biggest yields found
for extractions fluctuates from 78,7% to 82% [47, 48,
49]. Nonetheless, the total amount extracted from a
singular tissue is not enough for the market demand,
which implies the sacrifice of numerous organisms [42].
Thanks to the difficulties of source and extraction
procedure, the hyaluronidase is one of the most
expensive enzymes found in the market. As the medical
implications are constantly growing, several studies
attempted to use frozen seminal fluids from humans [47]
or optimize Streptococcus production [40] with no
success.
Otherwise, as it was mentioned before,
inhibition is mostly made with compounds obtained
from natural sources or its derivatives, so this aspect is
the most ethical aspect when working with
hyaluronidase in different stages such as investigation
or application in some industries like medical and
aesthetics, however, this does not mean some inhibitors
have origin on pharmaceutical drugs or on animals’
fluids, nevertheless these are also the inhibitors with
10
lower yields giving enough reasons for researchers and
investigators on not to use them and allowing to
conclude the reasons why they are not commonly used
in the applications on industries [44, 61].
Most common use of hyaluronidase is a reversal
agent in bad hyaluronic acid injection; at the beginning
it was difficult to understand that injection of hyaluronic
acid could had side effects as it is well received by the
skin because it is naturally produced by the dermis but
when the acid is injected in the deep dermis it starts
creating masses capable of roll against the bone showing
up physically as shadows and deformities and to reduces
the measure of the masses hyaluronidase is used but it
has to be carefully use because higher concentrations
can create side effects like predisposition to the
development of diseases related with tumors [70].
Hyaluronidase cannot be injected in its purest form
because it can interfere with skin hydration processes,
with the appearance of wrinkles and spots and with
growth of tumors or masses, for that reason the
recommend treatment of wrong hyaluronic acid
injection is a solution of hyaluronidase (70 U) and 0.5%
lidocaine with epinephrine (1.5 cc) in women of slim
build and average height between 30 and 40 years old
[71]; the injection of a solution of hyaluronidase as a
treatment is well received by the human body because
its activity gradually decrease for being involved in
processes like dilution, diffusion and deactivation [72].
Additionally, the hyaluronidase should be
injected following some security protocols to avoid side
effects. These protocols are kind of dependent of the
surgeon or esthetician, but in general terms is important
to dosage according to the quantity of affected tissues to
it be enough to allow a vessel that cannot longer
transport blood but not too much to foster an
environment of side effects, so in a nutshell it should be
a flooding of the zone but not an excess of the enzyme.
The injection of appropriate concentration and doses of
hyaluronidase depends on the surgeon experience and
comes along with some techniques such as the
identification of low or high-volume events, source of
hyaluronidase and decision making about injection, that
can be intra-arterial or externally applied and the
consequences of each of them [73].
Once the dermal procedure has been performed,
it is recommended that antibiotics be started for the first
two weeks [74]. In more cases, such as when large
amounts of hyaluronic acid filler must be removed, an
incision is recommended for drainage of the HA along
with the enzyme [75]. When hyaluronidases fail to
dissolve inflammatory nodules or long-lasting fillers,
steroids could be employed as an alternative [76]. For
these cases, they should only be administered after the
process with antibiotics has been initiated, as these
should only be applied when HYAL are not effective in
removing these fillers, i.e., as a second option and only
and exclusively for inflammatory nodules or long-
lasting fillers [74].
The procedure that is usually carried out when
performing HA dermal filler correction with
hyaluronidase relies on a very thorough analysis of the
area to be intervened. The area should be investigated
with ultrasonography prior to any treatment to identify
the amount, depth and extent of HA injected [77]. This
injection is expected to be as precise as possible and
11
only limited to the affected areas. The specialist should
adjust the amount of HYAL to be injected according to
the type of procedure performed, as fillers with higher
concentrations of HA will require more HYAL [78]. In
addition, it should be specified that the patient has not
ingested prior to the intervention, any of the antagonists
of these enzymes, such as anti-inflammatory agents,
among which are indomethacin, dexamethasone, and
salicylates; drugs of vegetable origin (flavonoids and
antioxidants), antihistamines, mast cell stabilizers,
heparin, and Vitamin C [79].
Ischemia is one of the side effects of HA dermal
fillers, it consists in the decrease of blood flow in the
tissues, generating a decrease of oxygen and nutrients in
the affected area, which can cause difficulty in
breathing, paralysis, or arrhythmia, depending on the
affected tissue or organ [80]. For this case it is necessary
to act quickly, and it is recommended to administer
HYAL within the first four hours after the intervention
[81]. There have been cases of patients who experienced
vision loss after being injected with these fillers, and
after instant HYAL administration, got recovered. [78].
DeLorenzi proposed a new treatment to avoid these
secondary effects, which involves dosing HYAL in
relation to the volume of the ischemic tissue. It consists
in performing repeated doses every hour to keep
hyaluronidase concentrations high in the affected area
and thus optimize the degradation of injected HA [82].
The figure 6 shows the diffusion of hyaluronidase
through the arterial wall.
 Fig 6. Diffusion of hyaluronidase through the arterial wall to degrade hyaluronic acid filler. [73]
The rate at which HYAL diffuses through the
arterial wall depends on the concentration administered.
From a clinical point of view, low concentrations are
much less effective, whereas high concentrations of
HYAL may result in higher intra-arterial concentrations
and, consequently, more efficient hydrolysis of HA
[73].
Therefore, an exhaustive process must be done
before the injection to determine the concentration of
hyaluronidase to inject and avoid possible allergic
reactions and side effects that may occur as mentioned
before.
Conclusion
It has been possible to identify different
hyaluronidase inhibitors, some better than others, but
unfortunately none of them follows the Michaelis –
Menten kinetic model making the inhibition more
difficult to understand. Among the best inhibitors, the
structural coincidence is that they all have a wide variety
of aromatic rings or carbonyl groups, and in general
terms they have shown a decrease in the synthesis of N-
acetylglucosamine producing a higher percentage of
inhibition. Plus, to date the best inhibitor found is
Vitamin C but unfortunately it is not known which of its
derivatives carries out the inhibitory activity or whether
12
the inhibitor functions as a “team player” in the
inhibition. Additionally, it has not been possible to
determine if the inhibition of hyaluronidase is carried
out for molecules with specific characteristics like
solubility; an example of this is the comparison between
oleanolic acid and vitamin C, the first one is an
amphipathic molecule and the second one is completely
soluble in water, but both showed a high inhibition
percentage, not allowing to determine if only one kind
of solubility could be used to inhibit the enzyme.
Until the moment of this review, it is
recommended to continue investigating the inhibition of
hyaluronidase now considering that it is involved in
various treatments and diseases and study the inhibition
from vitamin C derivatives, so it is possible to identify
the real fraction that makes this compound such a good
inhibitor.
In ethical terms it is important to resalt that
hyaluronidase is present in a lot of animals’ fluids, but
its extraction is difficult to get because the tissue most
be fresh and alive, so this includes an unethical method
of extraction because of animals’ exploitation. When
hyaluronidase is taken from human fluids it is also
counterproductive because of the specific conditions of
the tissue, plus the yields obtained are not higher enough
to use it as an object of investigation or in industrial
applications even when purifications are made.

When it is about industrial applications is

important to consider that hyaluronidase can generate
allergies and side effects such as tumor growth when the
concentrations used are higher than the recommended
or when the enzyme is purer than the recommendation
of dermatologists. To avoid the treatments on skin with
hyaluronidase the hyaluronic acid filler most be
evaluated before injection as well as the concentrations
to be injected, this will depend on the patient’s
characteristics and the state of its skin.

13
References
[1] T. Naranjo, R. Noguera-Salvá, and F. Guerrero, La matriz extracelular: morfología, función y bioseguridad (parte I). 2009.
[2] BA. Buhren, H. Schrumpf, NP. Hof, et al., Hyaluronidase: from clinical applications to molecular and cellular mechanisms.
2016
[3] F. Sabeh, R. Shimizu-Hirota and SJ. Weiss, Protease-dependent versus -independent cancer cell invasion programs:
threedimensional amoeboid movement revisited. 2009.
[4] T. Lammermann, BL. Bader, SJ. Monkley, T. Worbs, R. Wedlich-Soldner, K. Hirsch, et al., Rapid leukocyte migration by
integrin-independent flowing and squeezing. 2008.
[5] A.J. Kolarsick, M.A. Kolarsick and C. Goodwin, Anatomy and physiology of the skin. 2011.
[6] L. Juhlin, Hyaluronan in skin. 1990.
[7] J. Necas, L. Bartosikova, P. Brauner and J. Kolar, Hyaluronic acid (hyaluronan): a review. 2008.
[8] AB. Csoka, GI. Frost and R. Stern, The six hyaluronidase-like genes in the human and mouse genomes. 2001.
[9] B. Rzany, P. Becker-Wegerich, F. Bachmann, R. Erdmann and U. Wollina, Hyaluronidase in the correction of hyaluronic acid-
based fillers: a review and a recommendation for use. 2009
[10] NS. El-Saforya, AE. Fazaryb and CK. Leea, Hyaluronidases, a group of glycosidases: current and future perspectives. 2010.
[11] M. Meszaros, A. Kis, L. Kunos, A. Domonkos, D. Laszlo, Z. Lazar and A. Bikov, The role of hyaluronic acid and
hyaluronidase-1 in obstructive sleep apnoea. 2020.
[12] AL. Dunn, JE. Heavner, G. Racz, M. Day, Hyaluronidase: a review of approved formulations, indications and offlabel use in
chronic pain management. 2010.
[13] H. Garzón, Hyaluronic Acid. 2021
[14] C. DeLorenzi, Transarterial degradation of hyaluronic acid filler by hyaluronidase. 2014.
[15] C. Guerra, C. Rebello, B. Ribeiro, B. de Freitas, F. Costal, S. Stransky, C. Fonseca, D. Campolina, P. Pereira, R. Lira-da-Silva,
R. Machado, E. Kalapothakis and C. Chávez, Determination of hyaluronidase activity in Tityus spp. Scorpion venoms and its
inhibition by Brazilian antivenoms. 2019.
[16] K. Tomohara, T. Ito, S. Onikata, A. Kato and I. Adachi, Discovery of hyaluronidase inhibitors from natural products and their
mechanistic characterization under DMSO-perturbed assay conditions. 2017.
[17] U. Kuppusamy, H. Khoo and N. Das, Structure-activity studies of flavonoids as inhibitors of hyaluronidase. 1990.
[18] B. Arora, Anti-aging medicine.2008.
[19] C. De Jaeger, Fisiología del envejecimiento. 2018.
[20] V. Lobo, A. Patil, A Phatak and N. Chandra, Free radicals, antioxidants, and functional foods: Impact on human health. 2010.
[21] M.G. Rico, D. Oliva and G.B. Vega, Envejecimiento: algunas teorías y consideraciones genéticas, epigenéticas y ambientales.
2018.
[22] H. Vázquez and D. Asz, Hialuronidasa: aplicaciones dermatológicas. 2011.
[23] L. Duque, Efecto protector de plantas medicinales y aromáticas sobre componentes de la matriz extracelular: un enfoque en
la fotoprotección de la piel. 2016.
[24] J.M. Alcolea, P. Cornejo and M.A. Trelles, Perspectivas en el uso de materiales de relleno inyectables para tejidos blandos,
desde nuestra experiencia. 2ª parte. 2012.
[25] E. Bala, R. Hazarika, P. Singh, M. Yasir and R. Shrivastava, A biological overview of Hyaluronidase: A venom enzyme and its
inhibition with plants materials. 2018.
[26] A. Benitez, T. Yates, L. Lopez, W. Cerwinka, A. Bakkar and V. Lokeshwar, Targeting Hyaluronidase for Cancer Therapy:
Antitumor activity of Sulfated Hyaluronic Acid in Prostate Cancer Cells. 2011.

14
[27] H. Bouga, I. Tsouros, D. Bounias, D. Kyriakopoulou, M. Stavropoulos, N. Papageorgakopoulou, D. Theocharis, D. Vynios,
Involvement of hyaluronidases in colorectal cancer. 2010.
[28] C. McAtee, J. Barycki and M. Simpson, Emerging Roles for Hyaluronidase in Cancer Metastasis and Therapy. 2014.
[29] H. Siiskonen, M. Poukka, K. Tyynela-Korhonen, R. Sironen and S. Pasonen-Seppanen, Inverse expression of hyaluronidase 2
and hyaluronan synthases 1-3 is associated with reduced hyaluronan content in malignant cutaneous melanoma. 2013.
[30] S. Ohnuma, K. Miura, A. Horii, W. Fujibuchi, N. Kaneko, D. Osamu, H. Nagasaki, T. Mizoi, N. Tsukamoto, T. Kobayashi, M.
Kinouchi, M. Okabe, H. Sasaki, K. Shiiba, K. Miyagawa and I. Sasaki, Cancer-associated splicing variants of the CDCA1 and
MSMB genes expressed in cancer cell lines and surgically resected gastric cancer tissues. 2009.
[31] S. Langevin, D. Koestler, B. Christensen, R. Butler, J. Wiencke and H. Nelson, Peripheral blood DNA methylation profiles are
indicative of head and neck squamous cell carcinoma: An epigenome-wide association study. 2012.
[32] S. Patel, P. Turner, C. Stubberfield, E. Barry, C. Rohlff, A. Stamps, K. Tyson, J. Terret, G. Box, S. Eccles and M. Page,
Hyaluronidase gene profiling and role of HYAL-1 overexpression in an orthoptic model of prostate cancer. 2002.
[33] N. Swathi, K. Srikanth and S. Venipriya, Does the addition of hyaluronidase improve the quality of peribulbar anesthesia in
cataract surgery? – A randomized double blinded study. 2018
[34] M. Abu, M. Samir, M. Mohamed and M. Abdelhameed, The effect of adding cisatracurium versus hyaluronidase to
levobupivacaine and lidocaine mixture in single injection peribulbar block for cataract surgery. 2017.
[35] S. Chaand and A. Lahiri, Use of Hyaluronidase in Plastic Surgery: A review. 2021.
[36] C. Duan, J. Jimenez, C. Goergen, A. Cox, P. Sivasankar and S. Calve, Hydratation state and Hyaluronidase Treatment
Significantly Affect Porcine Vocal Fold Biomechanics. 2021.
[37] F. Ferreira, J. Marília, E. Suchi, M. Cardoso and P. Ferreira, Pharmacogenetic effects of angiotensin-converting enzyme
inhibitors over age-related urea and creatinine variations in patients with dementia due to Alzheimer disease. 2016.
[38] D.H. Pashley, F.R. Tay, C. Yiu, M. Hashimoto, L. Breschi, R.M. Carvalho and S. Ito, Collagen degradation by Host-derived
enzymes during aging. 2004.

[39] S. Braun, New Inhibitors of bacterial hyaluronidase - Synthesis and structure-activity relationships. 2014.
[40] M. Freeman, P. Anderson, M. Oberg and A. Dorfman, Preparation of purified hyaluronisade from bovine testis. 2011.
[41] K. Girish, D. Jagadeesha and K. Kemparaju, Snake venom hyaluronidase: an evidence for isoforms and extracellular matrix
degradation. 2018.
[42] C. Joyce, R. Jeyendran and L. Zaneveld, Release, extraction, and stability of hyaluronidase associated with human
spermatozoa. Comparisons with the rabbit. 2013.
[43] K. Kemparaju and K. Girish, Snake venom hyaluronidase: a therapeutic target. 2017.
[44] N. Kumar, N. Maity and P. Kumar, Matrix metalloproteinase, hyaluronidase and elastase inhibitory potential of standardized
extract of Centella asiatica. 2012.
[45] N. Mahesh, S. Balakumar and R. Vivek, Optimization and Production of Hyaluronidase by Streptococcus mitis MTCC 2695.
2012.
[46] H. Moawia and A. Fadl, Studies on Hyaluronidase Extracted from Staphylococcus aureus Isolated in Khartoum/Sudan. 2019.
[47] M. Oguzhan, O. Arslan and O. Guler, A new affinity method for purification of bovine testicular hyaluronidase enzyme and an
investigation of the effects of some compounds on this enzyme. 2014.
[48] W. Perloff and J. Nodine, The Extraction of Hyaluronidase from Human Testicles and Seminal Fluid. 2012.
[49] R. Sutti, M. Tamascia, S. Hyslop and T. Alves, Purification and characterization of a hyaluronidase from venom of the spider
Vitalius dubius (Araneae, Theraphosidae). 2014.
[50] Y. Tam and E. Chan, Purification and characterization of hyaluronidase from oral Peptostreptococcus species. 2013.
[51] C. Yang and P. Srivastava, Purification and properties of hyaluronidase from bull sperm. 2011.
[52] J. Remacle, C. Michiels and M. Raes, The importance of antioxidant enzymes in cellular aging and degeneration. 1992.
[53] K. Arunkumar, R. Raj, R. Raja and I. Carvalho, Brown seaweeds as a source of anti-hyaluronidase compounds. 2021.
[54] G. Pilco, D. Vinueza, K. Acosta and A. Torres, Actividad inhibitoria de la hialuronidasa del extracto de Piper Peltatum.
2019.
[55] T. Morikawa, Y. Nakanishi, N. Inoue, Y. Manse, H. Matsuura, S. Hamasaki, M. Yoshikawa, O. Muraoka and K. Ninomiya,
Acylated iridoid glycosides with hyaluronidase inhibitory activity from the rhizomes of Picrorhiza kurroa Royle ex Benth. 2020.
[56] S. Chambial, S. Dwivedi, K. Kant, P. John and P. Sharma, Vitamin C in Disease Prevention and Cure: An Overview. 2013.
[57] M. Spickenreither, S. Braun, G. Bernhardt, S. Dove and A. Buschauer, Novel 6-O-acylated vitamin C derivatives as
hyaluronidase inhibitors with selectivity for bacterial lyases. 2006.
[58] I. Tokeshi, T. Yoshimoto, N. Muto, S. Nakamura, K. Ashizawa, T. Nakada and H. Tatemoto, Antihyaluronidase action of
ellagic acid effectively prevents polyspermy as a result of suppression of the acrosome reaction induced by sperm-zona interaction
during in vitro fertilization of porcine oocytes. 2007.
[59] M. Asada, M. Surgie, M. Inoue, K. Nakagomi, S. Hongo, K. Murata, S. Irie, T. Takeuchi, N. Tomizuka and S. Oka, Inhibitory
effect of alginic acids on hyaluronidase and on histamine release from mast cells. 1997.
[60] K. Lee, J. Cho, E. Park and J. Choi, Anti-elastase and anti-hyaluronidase of phenolic substance from Areca catechu as a new
anti-ageing agent. 2001.
[61] H. Kakegawa, H. Matsumoto and T. Satoh, Inhibitory effects of some natural products on the activation of hyaluronidase and
their Anti-allergic Actions. 1992.
[62] A. Kaessler, M. Nourrisson, M. Duflos and J. Jose, Indole carboxamides inhibit bovine testes hyaluronidase at pH 7.0 and
indole acetamides activate the enzyme at pH 3.5 by different mechanisms. 2008.
[63] M. Oguzhan, O. Arslan and O. Ozensoy, A new affinity method for purification of bovine testicular hyaluronidase enzyme and
an investigation of the effects of some compounds on this enzyme. 2014.
[64] L. Zhang and M. Mummert, Development of fluorescent substrate to measure hyaluronidase activity. 2008.
[65] K. Mio and R. Stern, Inhibitors of hyaluronidases. 2002.
[66] H. Kakegawa, H. Matsumoto and T. Satoh, Inhibitory Effects of Hydrangenol Derivatives on the Activation of Hyaluronidase
and their Antiallergic Activities. 1998.
[67] F. Sánchez, C. Bosch and J.M. Cano, Spectrophotometry | Biochemical applications. 2019.
[68] O. Okorukwu and K. Vercruysse, Effects of Ascorbic Acid and Analogs on the Activity of Testicular Hyaluronidase and
Hyaluronan Lyase on Hyaluronan. 2003.
[69] A. Botzki, D. Rigden, S. Braun, M. Nukui, S. Salmen, J. Hoechstetter, G. Bernhardt, S. Dove, M. Jedrzejas and A. Buschauer,
L-Ascorbic Acid 6-Hexadecanoate, a Potent Hyaluronidase inhibitor. 2004.
[70] H. Jung, Hyaluronidase: An overview of its properties, applications, and side effects. 2020.
[71] V. Lambros, The use of hyaluronidase to reverse the effects of hyaluronic acid filler. 2004.
[72] D. Jiang, J. Liang and PW. Noble, Hyaluronan in tissue injury and repair. 2007.
[73] C. DeLorenzi, New High Dose Pulsed Hyaluronidase Protocol for Hyaluronic Acid Filler Vascular Adverse Events. 2017.
[74] 17 Murad Alam, Rosemara Hughart, Amelia Geisler, et al. Effectiveness of Low Doses of Hyaluronidase to Remove Hyaluronic
Acid Filler Nodules. 2018.
[75] 18 Selda Pelin Kartal. Is hyaluronidase injection effective in treating tear trough hyaluronic acid filler deformity?. 2018.
[76] 13 Kim DW, Yoon ES, Ji YH, Park SH, Lee BI, Dhong ES. Vascular complications of hyaluronic acid fillers and the role of
hyaluronidase in management. 2011
[77] 11 Cavallini M, Gazzola R, Metalla M, et al. The Role of Hyaluronidase in the Treatment of Complications From Hyaluronic
Acid Dermal Fillers. 2013.
[78]19 Claudio DeLorenzi. New High Dose Pulsed Hyaluronidase Protocol for Hyaluronic Acid Filler Vascular Adverse Events.
2017
[79] 22 Steinsapir KD. Treating filler related visual loss: Dermatol Surg. 2016
[80] Columbia Neurosurgery.Cerebral Ischemia - Symptoms, Treatment, Recovery. 2018.
[81] Cohen BE, Bashey S, Wysong A. The Use of Hyaluronidase in Cosmetic Dermatology: A Review of the Literature. J Clin
Investigat Dermatol. 2015
[82] Park TH, Seo SW, Kim JK, Chang CH. Clinical experience with hyaluronicacid-filler complications. 2016.