Hyaluronic Acid and ECM - Interplay and Medicine

#### **Chapter 6**

## Hyaluronic Acid Fillers: Where We Have Been and Where We Are Going

*Alexander Daoud and Robert Weiss*

#### **Abstract**

Since the approval of the United States' first hyaluronic acid (HA) filler in December 2003, HA fillers have become mainstays of soft tissue augmentation due to their favorable safety profile and minimally invasive treatment nature. The past two decades have not only brought an expansion in the popularity of HA fillers, but also in the number of available HA filler products and indications for cosmetic enhancement. Accordingly, HA filler injection has become one of the most commonly performed cosmetic procedures worldwide. The progression of HA filler products is a study in both biomedical engineering advancements, as well as evolving concepts of beauty and cosmesis. In this chapter, we review the history of these products, including their composition and indications for use. We then explore the prospect of HA fillers for the future of esthetic medicine, as they remain a vital component of nonsurgical soft tissue augmentation.

**Keywords:** soft tissue augmentation, biomedical engineering, cosmetic dermatology, hyaluronic acid

#### **1. Introduction**

Although typically thought of as outgrowths of modern medicine, interventions for esthetic and cosmetic purposes are well documented through multiple civilizations across history. In the non-surgical arena, soft tissue augmentation rapidly accelerated with the first reports of autologous fat transfer in the late 19th century; concomitantly, reports began to emerge from Europe on the use of injectable paraffin for rhytide reduction and soft tissue rejuvenation [1]. Shortly after the spread of paraffin injections across Europe and Asia, reports of embolic complications, as well as late-onset granulomatous reactions (later called 'paraffinomas') led to their eventual removal from the realm of esthetic practice, although non-medical/illicit injection of paraffin-containing substances still remain a sporadic issue in clinical practice today.

It was not until the 1940s that a new injectable agent found widespread use. Silicone, a dimethylsiloxane polymer, first entered clinical practice in Japan as an agent for breast augmentation. Over the following twenty years, silicone found widespread popularity across the United States, with the Dow Corning Corporation developing an injectable form of the material in the mid-1960s. As with paraffin decades prior, reports began to emerge in the 1960s–70s of delayed granulomatous

reactions to injected silicone. Termed *siliconomas*, these entities displayed a similar inflammatory pattern to paraffin, although migration of silicone due to the effects of gravity often led to granuloma formation at a site inferior or distal to the original site of injection [2]. As with paraffin, illicit silicone injection remains an issue across the world today, and clinicians should be mindful of this entity in the evaluation of granulomatous reactions following non-medical cosmetic treatments.

In 1981, the cosmetic landscape advanced with the United States Food and Drug Administration's (FDA) approval of Zyderm™, the first injectable filler approved for facial cosmetic enhancement. Derived from bovine collagen, Zyderm™ was comprised of a matrix of type I and type III collagen, which later formulations (Zyplast™) cross-linked with glutaraldehyde in order to slow the degradation of injected material [2]. Unlike the relatively inert nature of both paraffin and silicone, bovine collagen poses a significant risk of hypersensitivity reactions (3–3.5%, per population estimates) due to cross-speciation. Accordingly, skin testing prior to injection was necessary for all patients. Furthermore, although the collagen used for injection was derived from a closed and closely surveilled group of bovines, public concern regarding bovine spongiform encephalopathy ("mad cow disease") led to its fall from favor across the following decades [2].

The modern cosmetic injectables revolution found its genesis in 2003, when the FDA approved Restylane™, its first hyaluronic acid (HA) product. While other non-HA fillers were approved by the FDA for use in the following years – poly-Llactic acid (Sculptra™), polymethylmethacrylate (Belafill™), and Radiesse™ (calcium hydroxylapatite) among the most commercially successful – HA fillers have risen as some of the most popular agents available for nonsurgical facial cosmesis.

#### **2. Hyaluronic acid fillers: derivatives and bioengineering**

Hyaluronic acid is a glycosaminoglycan, a type of acid mucopolysaccharide that demonstrates significant hydrophilicity and serves as a key portion of the extracellular matrix of all organisms. In skin, HA is a key component of "ground substance", the acellular material found in the extracellular environment around collagen bundles of the dermis. In its structural support of collagen and elastin fibers, HA's main role in skin is to lubricate these extracellular structures by attracting water, which in turn produces a volumizing effect in the skin [3].

With time, the amount of HA in the dermis begins to diminish; accordingly, a hallmark of aging skin is a loss of both volume and elasticity. To this effect, HA fillers rose as a safe and logical solution for facial soft tissue enhancement: by reinstating the HA balance of the dermis, HAs draw water into the extracellular environment and lead to significant improvements in rhytide and soft tissue [2, 4]. Furthermore, due to HA's ubiquitous nature as a 'native component' of skin, the immunogenicity of HA filler products remains extremely low.

Engineering of HA fillers is possible through two broad methods: animalderived and bacterial-derived HAs. In the animal-derived group, HA is extracted from rooster combs and used for cosmetic injection [5]. Among the most successful in this group is Hylaform™, although animal-derived HAs have fallen from popularity due to their relatively shorter duration of effect, as well as their slightly increased immugenicity as compared to bacterial-derived HAs [1, 2]. In the bacterial-derived group, HAs are typically generated by fermentation of nonanimal stabilized hyaluronic acid (NASHA) from streptococcal species (Restylane™, Juvederm™, Captique™, Hydrelle™).

Two unique variables that determine an HA filler's physical properties are gel particle size, as well as the degree of HA crosslinking present. This is best

#### *Hyaluronic Acid Fillers: Where We Have Been and Where We Are Going DOI: http://dx.doi.org/10.5772/intechopen.97264*

exemplified by the Restalyne™ and Juvederm™ line of HA fillers, where adjustments in these two variables are used to create novel products with different properties and clinical applications. While many other HA filler products are commercially available, further analysis of this line of HA filler agents is chosen due to their ubiquitous presence in cosmetic offices worldwide.

Alterations in gel particle size are a hallmark of the Restylane™ family of products: products featuring particles of lower molecular weight (350 μm for Restylane™ versus 800-900 μm for Restylane™Lyft [formerly known as Perlane]) allow for a higher density of gel particles per unit volume (100,000 particles/ milliliter for Restylane™ versus 8,000 particles/milliliter for Restylane™Lyft). This is engineered through a process of sieving – by filtering particles mechanically, products are created with gel particles of a singular size [6]. Accordingly, while small gel particle-based products such as Restylane™, Restylane™-L, Restylane™ Kysse, and Restylane™ Silk have been approved for applications ranging from midface to lip and perioral augmentation, larger gel particle products such as Restylane™ Defyne, Restylane™ Refyne, and Restylane™ Lyft has been typically employed for deep rhytide correction and volumization of the cheeks and midface. Notably, Restylane™ Lyft also carries FDA approval for dorsal hand rejuvenation [1, 6, 7].

The effects of varying degrees of crosslinking are best displayed by the Juvederm™ family of products. Generally speaking, the greater the degree of crosslinking, the greater the degree of water absorption demonstrated by the filler; accordingly, more crosslinked products are typically used when significant volumizing or deep rhytide correction is desired. In contrast to Restalyne, which maintains the same concentration of hyaluronic acid throughout its product line, Juvederm™ products feature varying concentration of hyaluronic acid (24 mg/mL for Juvederm Ultra, Juvederm Ultra Plus, Juvederm Ultra XC, and Juvederm Ultra Plus XC; 20 mg/mL for Juvederm Voluma; 17.5 mg/mL for Juvederm Vollure; 15 mg/ mL for Juvederm Volbella). In addition, these products also differ from each other in both their percentage of HA crosslinking, as well as the manner in which they are crosslinked [8, 9].

Juvederm Ultra, Ultra Plus, and their XC variants feature a patented crosslinking technology termed Hylacross. With Hylacross, HAs of the same molecular weight are crosslinked together, creating a relatively uniform product of set viscosity and thickness. This is in contrast to Juvederm Voluma, Vollure, and Volbella, which use another patented technology called Vycross crosslinking. Vycross-generated fillers feature HAs of varying molecular weight, resulting in products that are reported to have a greater number of applications across the face, as well as a longer duration of action due to non-uniform degradation [8, 10].

Together, it is gel particle size and density, as well as degree/type of HA crosslinking, that influence the *G'*, or elastic modulus, of HA fillers. G' is a measure of a filler's response to shear or dynamic forces: the higher the G' of a filler, the greater its resistance to deformation or movement when under the influences of external force (such as dynamic facial movement). Accordingly, fillers with higher G' are often used for their volumizing and lifting effects, and deeper injections are needed [9, 11].

Emblematic of the role of G' in filler applications, Belotero Balance (Merz Esthetics) is a HA filler product that relies on a greater density of non-crosslinked HA in order to produce its intended effect. Generally speaking, the strength and volumizing effect of fillers is directly related to its degree of HA crosslinking – that is, free HAs are non-contributory to the strength, volumizing, or lifting potential of a filler product. Through a novel, patented formulation called Cohesive Polydensified Matrix – a process that generates a varying degree of crosslinked

HA in suspension with free HAs – a product is produced that features a significantly lower G' than other HA products (G' = 30 for Belotero Balance; G' = 545 for Restylane™ Lyft). Accordingly, Belotero ™ Balance is able to be used in both the intradermal and superficial subcuticular planes, with lower risk of the Tyndall effect (a gray-blue discoloration secondary to light scattering from superficially placed filler products) as compared to other HA fillers [9, 11, 12].

Depending on the product used, as well as the location being treated, HA fillers typically augment treated soft tissues for a period of 6–18 months. As compared to bovine collagen, hypersensitivity reactions are exceedingly rare, estimated at 1 in every 5000 injections. A notable benefit of these products, as compared to non-HA fillers, is their reversibility: if dissolution of filler is needed, injectable hyaluronidase may be used.

#### **3. Hyaluronic acid fillers: directions for the future**

Given their widespread success and popularity, HA fillers are likely to remain a mainstay in the cosmetic proceduralist's toolbox. Innovations in the field – ranging from methods of biochemical engineering to novel techniques for injection and treatment – all lend great promise to the future of HA fillers in esthetic practice.

In the realm of product development, one such advancement is demonstrated by Teosyal ™, an HA filler approved by the FDA in 2017. Through a novel patented synthesis method, a product with a lower bacterial protein and endotoxin load (as compared to other HA fillers) is generated, with a reported lower risk of potential hypersensitivity reactions as a result [9]. Another product generated through a unique method of synthesis is the Neauvia ™ family of HA fillers. Marketed as a 'fully organic' product, Neauvia ™ HAs are not synthesized from the streptococcal species typical of other HA fillers, but instead by *Bacillus subtilis*, a bacterium widely used in probiotic supplements. These HAs are then crosslinked with polyethylene glycol, creating a biocompatible hydrogel [13]. Lastly, Juvederm ™ has recently introduced Volux, a thicker HA with a concentration of 25 mg/mL, for lower face (jawline and chin) augmentation.

Continuing improvements in injection technique also offer a promising future of HA filler use. Blunt-tipped cannulas are rising in popularity as an alternative to hypodermic needles, as their use is associated with a statistically significant decrease in bruising, as well as lower pain associated with injection [14]. Additionally, cadaveric studies comparing blunt-tipped cannulas to sharp needles demonstrated a higher risk of intra-arterial injection with sharp needles, as well as a greater degree of filler extrusion across multiple anatomic planes (as opposed to one targeted level of injection) with needles as compared to blunt-tipped cannulas [15]. Accordingly, the use of cannulas has greatly advanced the technique, safety profile, and resultant cosmetic effect of HA fillers to areas such as the tear troughs and jawline, where anatomic plane precision is critical.

Exactness in volumetric dosing is highly dependent on injector experience, as elements such as HA filler viscosity, needle gauge, and plane/site of injection all influence the force needed to inject a bolus of filler agent. To elevate this aspect of treatment beyond volume marking on syringes and "injector feel" while introducing a bolus, Restylane ™ has recently introduced Skinboosters, a microdroplet HA treatment that uses a SmartClick® syringe to provide metered doses with each depression of the syringe plunger [16]. This technology has especially shown great promise when injecting into more superficial planes, as improper or non-uniform technique may otherwise result in nodule or 'lump' formation, as well as the Tyndall effect.

### **4. Conclusions**

Hyaluronic acid fillers are essential component of the cosmetic proceduralist's armamentarium. Their ubiquitous presence in clinics across the world, as well as their widespread acceptance by the general public, has made awareness of their use important for all clinicians. An understanding of their unique properties and physical features provides esthetic practitioners with a better comprehension of their treatment indications, potential complications, and treatment pitfalls, as well as an appreciation of developments on the horizon for future HA fillers.

### **Author details**

Alexander Daoud\* and Robert Weiss Maryland Dermatology, Laser, Skin, and Vein Institute, Baltimore, MD, USA

\*Address all correspondence to: adaoud@mdlsv.com

© 2021 The Author(s). Licensee IntechOpen. This chapter is distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/ by/3.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.

### **References**

[1] Kim JE, Sykes JM. Hyaluronic acid fillers: history and overview. Facial Plast Surg. 2011 Dec;27(6):523-528.

[2] Kontis TC, Rivkin A. The history of injectable facial fillers. Facial Plast Surg. 2009;25(2):67-72.

[3] Zerbinati N, Lotti T, Monticelli D, et al. *In Vitro* Evaluation of the Sensitivity of a Hyaluronic Acid PEG Cross-Linked to Bovine Testes Hyaluronidase. Open Access Maced J Med Sci. 2018;6(1):20-24.

[4] Matarasso SL, Carruthers JD, Jewell ML; Restylane Consensus Group. Consensus recommendations for soft-tissue augmentation with nonanimal stabilized hyaluronic acid (Restylane). Plast Reconstr Surg. 2006 Mar;117(3 Suppl):3S-34S; discussion 35S-43S.

[5] Kang DY, Kim WS, Heo IS, Park YH, Lee S. Extraction of hyaluronic acid (HA) from rooster comb and characterization using flow field-flow fractionation (FlFFF) coupled with multiangle light scattering (MALS). J Sep Sci. 2010;33(22):3530-3536.

[6] Khosravani N, Weber L, Patel R, Patel A. The 5-Step Filler Hand Rejuvenation: Filling with Hyaluronic Acid. Plast Reconstr Surg Glob Open. 2019;7(1):e2073.

[7] Dover JS, Rubin MG, Bhatia AC. Review of the efficacy, durability, and safety data of two nonanimal stabilized hyaluronic acid fillers from a prospective, randomized, comparative, multicenter study. Dermatol Surg. 2009 Feb;35 Suppl 1:322-330; discussion 330-1.

[8] Bogdan Allemann I, Baumann L. Hyaluronic acid gel (Juvéderm) preparations in the treatment of facial wrinkles and folds. Clin Interv Aging. 2008;3(4):629-634.

[9] Rohrich RJ, Bartlett EL, Dayan E. Practical Approach and Safety of Hyaluronic Acid Fillers. Plast Reconstr Surg Glob Open. 2019;7(6):e2172.

[10] Micheels P, Besse S, Sarazin D. Two Crosslinking Technologies for Superficial Reticular Dermis Injection: A Comparative Ultrasound and Histologic Study. J Clin Aesthet Dermatol. 2017;10(1):29-36.

[11] La Gatta A, Salzillo R, Catalano C, et al. Hyaluronan-based hydrogels as dermal fillers: The biophysical properties that translate into a "volumetric" effect. *PLoS One*. 2019;14(6):e0218287. Published 2019 Jun 11.

[12] Sundaram H, Fagien S. Cohesive Polydensified Matrix Hyaluronic Acid for Fine Lines. Plast Reconstr Surg. 2015 Nov;136(5 Suppl):149S-163S.

[13] Zerbinati N, Mocchi R, Galadari H, et al. *In Vitro* Evaluation of the Biological Availability of Hyaluronic Acid Polyethylene Glycols-Cross-Linked Hydrogels to Bovine Testes Hyaluronidase. *Biomed Res Int*. 2019;2019:3196723. Published 2019 Jun 12.

[14] Fulton J, Caperton C, Weinkle S, Dewandre L. Filler injections with the blunt-tip microcannula. J Drugs Dermatol. 2012;11(9):1098-1103.

[15] van Loghem JAJ, Humzah D, Kerscher M. Cannula Versus Sharp Needle for Placement of Soft Tissue Fillers: An Observational Cadaver Study. Aesthet Surg J. 2017 Dec 13;38(1):73-88.

[16] Lee BM, Han DG, Choi WS. Rejuvenating Effects of Facial Hydrofilling using Restylane Vital. Arch Plast Surg. 2015;42(3):282-287.

#### **Chapter 7**

## Hyaluronic Acid Derivatives for Targeted Cancer Therapy

*Nilkamal Pramanik and Sameer Kumar Jagirdar*

#### **Abstract**

Targeted therapeutics are considered next generation cancer therapy because they overcome many limitations of traditional chemotherapy. Cancerous cells may be targeted by various hyaluronic acid modified nanovehicles that kill these cells. Particularly, hyaluronic acid and its derivatives bind with high affinity to cell surface protein, CD44 enriched tumor cells. Moreover, these molecules have the added advantage of being biocompatible and biodegradable, and may be conjugated with a variety of drugs and drug carriers for developing various formulations as anticancer therapies such as nanogels, self-assembled and metallic nanoparticulates. In this chapter, we have covered various aspects of hyaluronic acid-modified delivery systems including strategies for synthesis, characterization, and biocompatibility. Next, the use of hyaluronic acid-modified systems as anti-cancer therapies is discussed. Finally, the delivery of small molecules, and other pharmaceutical agents are also elaborated in this chapter.

**Keywords:** Hyaluronic Acid, Nano-particulates, Immunogenicity, Biodegradation, Tumor Targeted delivery

#### **1. Introduction**

Nanoparticles have gained increased attention in the context of cancer therapy; however, the major challenge of targeting particles specifically to cancerous cells remains. Targeted delivery systems comprising cell-targeting ligands such as antibodies, peptides, folic acid, and various biomolecules have been developed to ensure tumor-specific delivery. One such targeting ligand is hyaluronic acid (HA).

HA, a glycosaminoglycan (GAG), is a natural polysaccharide present in the extracellular matrix of various soft connective tissues such as the vitreous humor, dermis of the skin, hyaline cartilage, and synovial fluid of the body. It is watersoluble, viscoelastic, biodegradable, biocompatible, and non-immunogenic [1–4]. HA is a polyanionic mucopolysaccharide consisting of β-1,3 and β-1,4 glycosidic bonds between repeating units of D-glucuronic acid and N-acetyl-D-glucosamine [5].

As an intrinsic part of the ECM, HA participates in different biological functions of the cell, including signal transduction, vascularization, cell migration, and tissue remodeling as schematically represented in **Figure 1**. Additionally, the presence of modifiable hydrophilic functional groups hydroxyl, carboxyl, and N-acetyl increases its potential as an adaptable system for the delivery of proteins, nucleic acids, and anti-cancer agents by grafting or modification with different nanoparticles. Based on cellular interaction studies, HA has emerged as a tumor-targeting agent in cancer therapy. It exhibits a

**Figure 1.**

*Schematic representation of physico-chemical properties and functions of hyaluronic acid (HA) in the native tissue environment.*

high binding affinity towards the CD44 cluster [6], which is over-expressed in numerous malignant cancer cells [7–9].

Considering its hierarchical structure and potential as a targeting agent, HA can be modified using small biomolecules [10], nanotubes [11], or different types of metal and non-metal nanoparticles [12] in various formulations. HA modified particles are effectively ingested by cells, and HA is enzymatically degraded intracellularly [13], resulting in the delivery of only the particles and their cargo inside the cell.

This chapter covers various aspects of HA-modified delivery systems, including strategies for synthesis, characterization, and biocompatibility. Furthermore, the characteristics of different delivery systems such as nanogel, micelle, liposome, and metallic or non-metallic nano-particulates are discussed. The mechanism underlying the delivery of small molecules, nucleic acids, and other pharmaceutical agents (*in vitro*, *in vivo*, or clinical applicability) are also presented in this chapter.

#### **2. Hyaluronic acid–a primer**

HA was discovered in 1934 when scientists Karl Meyer and John Palmer isolated a new kind of polysaccharide from bovine vitreous humor, which was later termed as hyaluronan [14]. Hyaluronic acid is a polysaccharide composed of repeating units of β-1,3-N-acetyl-D-glucosamine and β-1,4-D-glucuronic acid linked by β-1,3 and β-1,4 glycosidic bonds. It is a component of the extracellular matrix and mediates various cellular functions. HA is a ligand for the cell surface receptor CD44. CD44

is a transmembrane protein that is typically expressed by a number of cells, but is over-expressed in different types of metastatic tumor cells, including those of brain, breast, prostate, colon, bladder, and head and neck cancers [7–9, 15–17]. It has a significant role in cell proliferation, migration, metastasis, and cell–cell and cell-matrix signal transduction [18].

#### **2.1 Synthesis**

Hyaluronic acid is usually obtained from different biological sources. The microbial synthesis pathway is preferred as it is cost-effective and an environmentally benign process. The gram-positive bacterium, *Streptococcus zooepidemicus* is used for large-scale production of HA via a fermentative pathway as shown in **Figure 2(A)** [19]. The biosynthetic pathway of HA is as follows: initially, glucose-6-phosphate is converted to uridine diphosphate glucose (UDP-glucose) in the presence of α-phosphoglucomutase and UDP-glucose dehydrogenase followed by UDP-glucose dehydrogenase assisted oxidation into UDP-glucuronic acid. Next, an amide group is transferred from glutamine-fructose-6-phosphate to fructose-6-phosphate, followed by rearrangement of the phosphate leading to the formation of glucosamine-1-phosphate. Subsequently, acetylation and conjugation of UTP to glucosamine-1-phosphate generates the second precursor of HA. In the final step, hyaluronan synthase polymerizes the two precursors to produce HA, which is presented as an extracellular capsule as confirmed by electron microscopy (see **Figure 2(B)**) [20]. However, the pathogenicity of the bacterium limits its use for mass production of HA and therefore, several recombinant strains such as *Agrobacterium* sp. ATCC 31749 and recombinant *Escherichia coli* have been adapted as an alternative source of HA production [21, 22].

#### **Figure 2.**

*(A) Hyaluronic acid biosynthetic pathway in* S. zooepidemicus*. Glucose is first converted to glucose-6-phosphate by hexokinase which then enters one of two distinct pathways to form UDP-glucuronic acid (*pgm, hasC *and* hasB*) or UDP-*N*-acetylglucosamine (*hasE, glmS, glmM *and* hasD*). These precursors are subsequently bound together via the action of hyaluronic acid synthase or HAVE (encoded by* hasA *in* S. zooepidemicus*) to form hyaluronic acid. [Ref. [19], reproduced with permission from publishing authority]. (B) Electron micrograph section of* Streptococcus equi *subsp.* Zooepidemicus *(*S. zooepidemicus*) cells obtained from the late exponential phase of an aerated bioreactor culture. Thin sections were stained with uranyl acetate and lead citrate and examined with a Jeol JEM-1010 transmission electron microscope at an accelerating voltage of 80 kV.*  Bar *1 μm. [Ref. [20], reproduced with permission from publishing authority].*

#### **2.2 Properties and uses**

As mentioned previously, HA is a polymer. The physiochemical properties such as rheology and viscoelasticity of the polymer depend on the length of HA. Previous studies have demonstrated that higher molecular weight HA has better wound healing properties and is more effective for orthopedic treatment whereas low molecular weight HA has a prominent role in angiogenesis and is an effective immuno-stimulant [23]. A variety of HA oligosaccharides have been synthesized chemically, ranging from disaccharides to hexasaccharides to improve biomedical availability and use. Lu et al. synthesized HA decasaccharides using a chemoselective glycosylation pathway [24]. Commercially available D-glucose and D-glucosamine hydrochloride are chemically connected in the presence of an activator to generate various HA-derivatives [25].

#### **3. Applications in cancer therapy**

On the basis of their physiochemical properties, different HA-based nanoformulations have been investigated for their therapeutic application in tumortherapy. HA conjugated drugs, polymers, and lipids may self-assemble in aqueous solvents, and this property has been used to synthesize a number of self-assembled nano-particulates that are either made of the drug or contain the drug.

#### **3.1 Drug-HA conjugates**

Direct conjugation of drug molecules with HA results in the development of systems that are not only capable of targeting but improve solubility as well as blood circulation times of the drug itself. Some of the methods to synthesize drug-HA nanoparticles are summarized in **Figure 3**, and below, we discuss a few examples of the use of HA in developing new anti-cancer therapeutics.

The first example, presented in **Figure 3(A)**, involves the conjugation of HA with succinic anhydride derived paclitaxel (2-succPTX, a tubulin inhibitor) using glutathione (GSH) sensitive cystamine (or non-sensitive adipic dihydrazide) as a cross-linker. The resultant self-assembled nanoparticles formed were analyzed using <sup>1</sup> H nuclear magnetic resonance (NMR), Fourier-transform infrared spectroscopy (FTIR), and UV–visible spectroscopy. Transmission electron microscopy (TEM) and atomic force microscopy (AFM) analysis demonstrated the presence of spherical shaped nanoparticles of diameter 150 nm. The nanoparticles accurately targeted cancer cells with significant anti-tumor efficacy both *in vitro* and *in vivo* as compared to free PTX [26]. Along similar lines, another HA-PTX nanovehicle showed excellent results in reducing tumor size with the increase of survival rate in an *in vivo* mouse xenograft model bearing ovarian cancer cells [27]. A recent development in this area was to improve the loading of PTX in these self-assembled nanoparticles through the use of dimethylsulfoxide (DMSO) and polyethylene glycol (PEG) in the organic phase. This nano-system was suggested to have a 10–20% increase in PTX loading, and as a result, had significant anti-tumor activity against the RT-4 and RT-112/84 bladder carcinoma cell-lines [28].

The second example is the synthesis of a HA-doxorubicin (DOX) based selfassembled pro-drug that formed spherical core-shell nanostructures (**Figure 3(B)**) of 180–200 nm diameter. It displayed good biocompatibility and pH-responsive controlled Dox release in a cervical cancer model and exhibited excellent tumor inhibitory effects [29]. An ion-pairing based Dox-HA nano-assembled structure

*Hyaluronic Acid Derivatives for Targeted Cancer Therapy DOI: http://dx.doi.org/10.5772/intechopen.97224*

#### **Figure 3.**

*(A) Synthesis of 2′-succinyalted paclitaxel (PTX) and cystamine (ss) modified hyaluronic acid (HA) g-paclitaxel (PTX) [HA–ss–PTX] through two consecutive pathways. Initially, PTX was functionalized with succinic anhydrate to obtain an active free carboxylic acid group which was further grafted with HA in the presence of cystamine as a cross-linker using EDAC. HCl and NHS reaction chemistry mechanism. [A(i&ii)] illustrate the schematic and TEM images of HA-PTX based self-assembled structure. [Ref. [26], adapted with permission from publishing authority]. (B) Schematic presentation of the synthesis of amide methyl 4 (aminomethyl) benzoate crosslinked with self-assembled HA-doxorubicin graft. (C) Synthesis of 2, 7-succinyalted cisplatin [2, 7-Succ-Pt(IV)] followed by ethylene diamine (EDA) mediated conjugation with HA using EDAC. HCl and NHS reaction chemistry to form HA-EDA-Pt(IV) pro-drug. [C(i&ii)] AFM images of HA–EDA–Pt (IV) nanoconjugate at an optimal dilution ratio, indicating the formation of microspheres with an average diameter of 200 nm. [Ref. [31], adapted with permission from publishing authority].*

with a liposomal delivery system displayed sustained intracellular release of Dox in CD44+ cancer cells with enhanced therapeutic efficacy in a mouse model [30].

The third and final example is the conjugation of HA with cisplatin. Cisplatin is an anti-cancer drug that causes adverse side-effects. Increasing cancer cell-specific intracellular delivery of cisplatin may reduce side effects, which might be possible by conjugating HA onto the drug. Ling et al. have demonstrated the synthesis of HA conjugated cisplatin using N-(3-dimethylaminopropyl)-N′-ethylcarbodiimide hydrochloride. The synthesized pro-drug was verified using 1 HNMR, 13C NMR, FT-NIR, AFM, and DSC analysis. The prepared pro-drug was spherical (**Figure 3(C)**) and showed CD44 mediated endocytosis with negligible stimulation to blood vessels. Systemic toxicity studies indicated that the drug was safe and actively delivered cisplatin to kill tumor cells with reduced adverse effect on healthy cells [31].

#### **3.2 Drug loaded HA based nanoparticles**

HA may also be conjugated with lipids and other small molecules, which also self-assemble to form particulates that may be used to encapsulate drugs. One example of such a system is deoxycholic acid conjugated with HA, which results

in the formation of micelles that may, in turn, be loaded with drug molecules (**Figure 4(A)**). In the specific study presented in this figure, Huo and colleagues showed that the average hydrodynamic size and colloidal stability in terms of 'zeta potential' of the resultant micelle was 120 nm and 36 mV, respectively. These particles were capable of releasing taxol (the drug here) into the cytoplasm of cancer cells, causing tumor apoptosis, with a minimum adverse effect on healthy cells [32]. In another study, cholesterol coupled HA was employed as the amphiphilic molecule that self-assembles into nanoparticles that encapsulate both the anticancer drug Dox and magnetic nanoparticles (**Figure 4(B)**). This multifunctional delivery system exhibited high cytotoxicity and cellular uptake against several cancer cell lines such as HeLa, HepG2, and MCF7 [33].

Utilizing HA-molecule conjugated self-assembled nanovehicles for drug loading enables the development of combinatorial therapeutic approaches. As an example of such an approach, the photo-sensitizer, Ce6, has been coupled with HA to form a self-aggregated system that is capable of delivering therapeutic drugs. Such a system was used in a human colon xenograft and displayed a significant anti-tumor effect

#### **Figure 4.**

*(A) Schematic illustration of cystamine (ss) cross-linked deoxycholic acid (DOCA) conjugated hyaluronic acid (HA) [HA-ss-DOCA conjugate] nanoparticles. (B) Schematic illustration of the synthesis of cholesterolconjugated HA (ch-HA) and the formation of DOX/SPION loaded ch-HA micelles. [Ref. [33], adapted with the permission from publishing authority]. (C) Schematic illustration of the preparation of HA-grafted micelles (HA-M) [Ref. [36], reproduced with permission from publishing authority]. (D) Preparation of HA/TN-CCLP and other liposomes. [Ref. [37], adapted with permission from publishing authority.*

in a mouse model [34, 35]. In the case of brain tumor therapy, the delivery of chemotherapeutic agents is hindered by the sophisticated blood–brain barrier (BBB) [36], and such combinatorial therapeutics could be beneficial for hard-to-treat cancers such as glioblastoma multiforme (GBM). Combinatorial chemotherapy strategies based on lauroyl-gemcitabine, honokiol (HNK), and HA grafted micelle formulations (**Figure 4(C)**) were shown to significantly suppress GBM in an *in vivo* xenograft model [36]. Similarly, Liposomes composed of TAT-NBD (TN, a 22 amino acid cell-penetrating peptide) modified HA, encapsulating celecoxib (CXB) and curcumin (CUR) (HA/TN-CCLP) (**Figure 4(D)**) were reported to block nuclear factor-κB (NF-κB) and signal transducer and activator of transcription 3(STAT3) signaling pathways, potentially inhibiting tumor growth and metastasis by improving infiltration of inflammatory cells [37].

#### **3.3 Gel formulations**

One of the major advantages of using HA is its ability to be used in diverse forms. As HA is an extracellular matrix protein, it may also be used to form hydrogels by itself or in combination with other polysaccharides. Injectable polymeric hydrogels have made a significant contribution to active targeted delivery of chemotherapeutic agents as the 3-dimensional porous environment allows for pH or thermo-sensitive controlled intracellular release of cargo. A HA modified chitosan grafted poly-N-isopropylacrylamide hydrogel was reported to have the loading capacity of Dox/folic acid-g-graphene sheets with high killing efficacy against MCF7 breast cancer cells. An *in vivo* study also demonstrated delivery of anti-tumor agents using the same system [38].

Interferon α-2a (IFN α-2a) loaded HA–tyramine hydrogels have also been shown to have anti-tumor effects, while native IFN α-2a injection did not show any anti-cancer effects. This was due to the controlled release of IFN α-2a from the hydrogel network [39]. Such gels may also be developed as nano-formulations, as demonstrated by Jaya Kumar and colleagues who showed that a redox-sensitive Dox loaded chitin-cystamine-HA nanogel may be used to specifically kill CD44+ HT-29 cells [40].

#### **3.4 Graphene oxide (GO) based formulations**

In the past couple of decades, carbonaceous compounds have gained significant attention in cancer therapy due to their large surface area and bio-sensing, bioimaging, cellular probing, and drug carrier abilities. Owing to its 2D structure, biocompatibility, and water-dispersion features, graphene oxide (GO) and its HA conjugate have been investigated as anti-cancer drug delivery platforms. Three specific examples are discussed here.

First is the development of HA and Arg-Gly-Asp (RGD) peptide coated graphene oxide as a nano-carrier for Dox. Raman spectroscopic analysis revealed two strong peaks at 1350 cm−1 and 1550 cm−1 due to the presence of D and G bands in exfoliated graphene oxide, as shown in **Figure 5(A)**, which was further confirmed by TEM. The nano-carrier was composed of a single transparent layer with a gauzelike lid layer. It exhibited high Dox loading capacity and excellent cytotoxicity when tested on an ovarian cancer cell line, SKOV-3. It was also found to be biocompatible when tested on a healthy human cell line, HOSEpiC [41].

The second is the development of a redox-sensitive near-infrared (NIR) controlled system consisting of both HA and GO. Yin et al. demonstrated (**Figure 5(B)**) that a Dox/HA-cystamine-GO nano-carrier displays selective targeting and glutathione responsive release of Dox in the cytosol without any collateral damage to healthy

#### **Figure 5.**

*(A) Schematic illustration of the preparation and characterization of RBITC labeled Q-graphene (HA-Q-G-RBITC)/DOX nanoparticles and HA-mediated endocytosis. (i) XPS analysis of Q-graphene (a), Q-graphene-COOH (b), Sulf-Q-graphene (c), and PEG Q-graphene (d). (ii) TEM image of Q-graphene. (iii) cytotoxic effects of Q-G-RBITC/DOX, HA-Q-G-RBITC/DOX, and DOX against A549 cells with increasing DOX concentration. The error bar represents standard deviation (n = 5). (iv) CLSM images of A549 cells incubated with (A) HA-Q-G-RBITC, (B) HAQ-G-RBITC/DOX, or (C) Q-G-RBITC/DOX for 5 h. (D) MRC-5 cells incubated with HA-Q-G-RBITC/DOX for 5 h. [Ref. [41], reproduced with permission from publishing authority]. (B) Synthesis of HSG-DOX nanosheets. (i) Raman absorption spectra of HCG, HSG, GO-COOH, and GO. (ii) In vitro DOX release from HSG-DOX after incubation with glutathione at 37°C. (iii) In vivo fluorescent imaging of MDA-MB-231 tumor-bearing nude mice at 4, 16, and 36 h after intravenous injection of Cy7-labeledHSG-DOX and HCG-DOX nanosheets with or without pre-injection of free HA at HSG/HCG dose of 5 mg/kg. (iv) tumor growth curves after intravenous injection of different formulations at a DOX dose of 5 mg/kg. \*\*P < 0.01. [Ref. [12], reproduced with permission from publishing authority]. (C) Synthesis of dox loaded magnetite nanoparticles/GO-HA. Cytotoxicity induced by combination of drug treatment and hyperthermia. GO-HA-iron oxide particulates (i) without or (ii) with dox were cultured with MDA-MB-231 cells and exposed to magnetic fields, and cell viability was measured. Cell viability measurements are normalized to control cultures of cells in the absence of particulates, drugs, and hyperthermia. Paired Student's t-test was performed to compare hyperthermia treatment to their respective no-heat controls. \*\*p < 0.01. [Ref. [43], adapted with permission from publishing authority].*

*Hyaluronic Acid Derivatives for Targeted Cancer Therapy DOI: http://dx.doi.org/10.5772/intechopen.97224*

cells. As compared to free Dox, the NIR irradiated nanosystem exhibited enhanced cytotoxicity in a xenograft tumor model [12]. A similar cytotoxic effect was observed when HiLyte 647 loaded nano GO-HA was used to treat melanoma. Photo-thermal treatment resulted in the complete ablation of tumor tissue without any further tumorigenesis [42].

The third example is our own work on a formulation containing magnetic nanoparticle decorated GO-HA, which was evaluated for magnetothermal and CD44 (+) positive breast cancer targeted cancer therapy. A shown in **Figure 5(C)**, the nanoplatform can be loaded with various types of chemotherapeutic agents such as Dox and Ptx. Furthermore, the study revealed that the nanoplatform had significant anti-tumor activity under magnetic hyperthermia in the MDA MB231 cell line. These nanovehicles provide a versatile platform for next-generation cancer therapy [43].

#### **3.5 Other carriers**

Owing to their high stability, biocompatibility, and tunable porous architecture, mesoporous silica nanoparticles (MSNPs) have been used as multifunctional tumor-targeting nano-carriers. Dox loaded HA modified MSNPs have been developed and tested against HCT-116 cells, as shown in **Figure 6(A)**. As part of the morphological analysis, TEM revealed spherical nanoparticles organized as a hexagonally packed mesoporous structure with a mean particle size of 70–100 nm. Surface modification of the MSNPs was confirmed by 13C NMR analysis. Strong absorption peaks at 43, 22, and 10 ppm and broad peaks at 70–180 ppm confirmed the presence of the methylene carbon in NH2-MSNPs and the anomeric carbon in HA, respectively. Compared to free Dox and Dox loaded MSNPs, Dox-HA-MSNPs exhibited a significantly greater anti-proliferative effect because of better CD44 mediated uptake of HA modified nanoparticles at physiological pH [44].

A similar approach was used to fabricate mesoporous silica nanoparticles, post-functionalized with PEG-PDS-NH2 [poly(poly(ethyleneglycol) methacrylateco-pyridyldithioethyl methacrylate-co-2 aminoethylmethacrylate], followed by HA decoration for selective targeting. As shown in **Figure 6(B)**, Dox loaded nanoparticles demonstrated clathrin and macropinocytosis-mediated cellular uptake with the killing of CD44 positive HeLa cells [45].

Gold nanoparticles (AuNPs) in cancer therapy have dual functionality due to the presence of a bioactive surface, contrast ability, and photodynamic features. Kang et al. developed HA conjugated pheophorbide-A coated AuNPs (**Figure 6(C)**) that had excellent colloidal stability and photoactivity in the intracellular environment [46]. Electron microscopy analysis of the hybrid nanomaterial revealed spherical nanoparticles with a mean diameter of 70–80 nm, whereas native gold nanoparticles had a mean diameter of 10–15 nm. The increase in size is attributed to the surface coating of the AuNPs. Active targeting was observed 48 h post-injection of PheoA-HA/AuNPs as indicated by bright fluorescence intensity at the tumor site rather than elsewhere, suggesting CD44 receptor-mediated accumulation of nanoparticles with minimum adverse effects on healthy tissues. *In vivo* study of administration of PheoA-HA/AuNPs revealed their excellent anti-tumor efficacy with 3-fold to 5-fold decrease in tumor size compared to free PheoA, saline, and AuNPs. In another study, Wang et al. synthesized {(Au0)100G5.NH2-FI-DOTA (Mn)-HA, where DOTA-1, 4, 7, 10-tetraazacyclododecane-1, 4, 7, 10-tetraacetic acid} NPs which facilitated selective internalization of dendrimers in tumor cells whose imaging capability is presented in **Figure 6(D)** [47].

Super-paramagnetic iron oxide nanoparticles have gained tremendous attention in targeted cancer therapy due to their diverse properties, including

#### **Figure 6.**

*(A) Synthesis of different hyaluronic acid (HA) modified metal nano formulations for targeted cancer therapy. [A(i&ii)]. 13CNMR spectra of NH2-MSNs and HA-MSNs. [iii(a&b)]. TEM images of MSNs and (iv) HA-MSNs. (D) Cytotoxicity of free dox, dox-HA-MSNs, dox-MSNs, HA-MSNs and MSNs againstHCT-116 cells at different concentrationsC1, C2andC3 (for details, see table v). [Ref. [44], adapted with permission from publishing authority]. (B) Schematic illustration of efficient mesoporous nanoparticle-mediated DDSs using noncovalent polymer gatekeepers and HA conjugation for targeting capability. (i) TEM images of HA-PMSNs, (ii) cumulative dox release profiles of 62 Mol% crosslinked PMSNs with (1 and 5 mM) and without GSH. (iii) cell viability analysis of HA-PMSNsCD44-positive in HeLa cells. [Ref. [45], adapted with permission from publishing authority]. (C) Schematic representation of the synthesis of {(Au0)100G5. NH2-FI-DOTA (Mn)-HA} NPs. (i) TEM image of the {(Au0)100G5.NH2-FI-DOTA (Mn)-HA} NPs. The scale bar in each panel measures 20 nm. (ii) In vivo CT images of orthotopic liver tumors at different times after a 0.3-mL intravenous injection of a {(Au0)100G5.NH2-FI-DOTA (Mn)-HA} NP solution (0.3 mL* 

#### *Hyaluronic Acid Derivatives for Targeted Cancer Therapy DOI: http://dx.doi.org/10.5772/intechopen.97224*

*in PBS, [Au] = 120 mM). (iii) In vivo MR images of orthotopic liver tumors at different times after an intravenous injection of 0.3 mL of a {(Au0)100G5.NH2-FI-DOTA (Mn)-HA} NP (300* μ*g Mn) solution in PBS. [Ref. [47], adapted the permission from publishing authority]. (D) Synthesis of HA-modified magnetite nanoparticles (HA-Fe3O4 NPs) (i) TEM micrographs of HA-Fe3O4 NPs. The viability of MIAPaCa-2 cells after treated with PBS, nHA-Fe3O4 NP and HA-Fe3O4 NPs at the different Fe concentrations for 24 h (ii) or 48 h (iii) at 37 oC by the CCK-8 assay (iv) In vivo transverse T2 MR images of tumors after intravenous injection of the nHA-Fe3O4 NP ((a) 7 days; (c) 14 days; (e) 21 days) and HAFe3O4 NPs ((b) 7 days; (d) 14 days; (f) 21 days) ([Fe] 1 mg/mL, in 200 mL saline) at different time points post i.v.-injection. (v) In vivo biodistribution of hearts, livers, spleens, lungs, kidneys, and tumors 24 h post intravenous injection of the nHA-Fe3O4 NP and HA-Fe3O4 NPs (600 mg Fe, in 0.3 mL PBS). (vi) The ability of MIAPaCa-2 cells to uptake PBS (a), nHA-Fe3O4 NP (b) and HA-Fe3O4 NPs (c) ([Fe] 50 mg/mL) 4 hours after treatment, MIAPaCa-2 cells treated with PBS were used as control, scale bar ¼ 10 mm. [Ref. [48], adapted with permission from publishing authority].*

biocompatibility, enabling their use as a contrast agent for MRI and in magnetothermal therapy. Moreover, their tendency for aggregation in an aqueous medium is avoided by coating with active biopolymers and cancer-targeting agents. For example, HA modified and fluorescein isothiocyanate decorated iron nanoparticles may be utilized as an efficient probe for targeted MRI assisted cancer therapy, as shown in **Figure 6(E)** [48].

Another use of these particulates could be targeted brain tumor therapy, and a specific example is the development of HA-polyethylene glycol stabilized magnetite nanoparticle modified nano-sized liposomes. Dox loaded versions of these nanoparticles were shown to enhance drug release under induced hyperthermia (43°C). Confocal microscopy and flow cytometry analysis revealed CD44 targeted internalization of liposome nanoparticles in glioblastoma (U87 cells) tumor cells with a dual effect i.e. magneto-thermal and chemotherapeutic triggered the killing of tumor cells *in vitro*, suggesting their potential role as next-generation *in vivo* anti-cancer nano-vehicles [49].

#### **4. Immunogenicity of HA-based nanoparticles**

The extent of interaction between nanoparticles and serum proteins, i.e. the formation of the protein corona, is a key factor deciding the intravenous delivery efficiency of the nanoparticles. The protein corona may be modified using coatings that alter the surface properties of the nanoparticles. Almalik et al. showed that modifying chitosan NPs with HA avoided inflammatory protein adsorption compared to nanoparticles not coated with HA [50]. Similarly, reactive oxygen species (ROS) production was suppressed when activated macrophages were treated with HA modified chitosan nanoparticles. The secretion of cytokines such as TNF-α and IL-1β were drastically reduced, indicating low immunogenicity of the HA-CS NPs without any collateral biological responses [51]. Zaki et al. also demonstrated that HA-chitosan NPs were taken up at a relatively slower rate compared to NPs not coated with HA. Moreover, the extent of internalization was two-fold lesser [52]. Further studies are required to verify that HA coatings decrease the immunogenicity of particulates.

#### **5. Conclusions**

Research in the development of active cancer-targeting agents led to the discovery of various cell surface molecules that control cellular function via different signaling pathways. CD44 is a cell surface protein that plays a significant role in tumorigenesis, metastasis, and proliferation of cancer cells. Several studies have demonstrated that the interaction of CD44 and HA, an extracellular component,

leads to the progression, growth, and metastasis of cancer cells via different signaling pathways. Consequently, strategies have been developed to fabricate HA mediated tumor targeting nanoplatforms. Moreover, it has been reported that conjugation of HA with different nanoparticles increases the internalization of therapeutic molecules via enhanced permeability and retention or CD44 mediated endocytosis with increased therapeutic efficacy both *in vivo* and *in vitro*. Particularly, various drug loaded targeting strategies have emerged, including redox, thermosensitive, and pH sensitive self-assembled HA-prodrug delivery and HA modified metallic and non-metallic nanovehicle-mediated delivery. In summary, HA-based nanoliposomes, micelles, and nano-carriers are promising therapeutic platforms for the delivery of multifunctional cargo in the context of active targeted cancer therapy, paving the way for next-generation clinical cancer therapy.

### **Acknowledgements**

Dr. Nilkamal Pramanik acknowledges DST, SERB- DST (NPDF), Govt. of India, for their financial support.

### **Conflicts of interest**

No conflict of interest is declared.

### **Author details**

Nilkamal Pramanik\* and Sameer Kumar Jagirdar Centre for BioSystems Science and Engineering, Indian Institute of Science, Bengaluru, Karnataka, India

\*Address all correspondence to: nilkamalorganic@gmail.com

© 2021 The Author(s). Licensee IntechOpen. This chapter is distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/ by/3.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.

*Hyaluronic Acid Derivatives for Targeted Cancer Therapy DOI: http://dx.doi.org/10.5772/intechopen.97224*

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#### **Chapter 8**

## Plant Natural Products: A Promising Source of Hyaluronidase Enzyme Inhibitors

*Muhammad Zeeshan Bhatti and Aman Karim*

#### **Abstract**

Hyaluronidase enzyme degrades hyaluronan, the primary component of the extracellular matrix found in connective tissues animals and on the surface of certain pathogenic bacteria. The degradation of hyaluronan is linked to a wide range of physiological and pathological process. Inhibiting the hyaluronidase enzyme is thus significant as an approach to treat a variety of diseases and health conditions such as anti-fertility, anti-tumor, antimicrobial, and anti-venom/toxin agents. HAase inhibitors of different chemical types have been identified include both synthetic compounds and constituents obtained from naturally sources. Plant natural products as HAase inhibitors are unique due to their structural features and diversity. Medicinal plants have historically been used as contraceptives, antidote for snakebites and to promote wound healing. In recent years, small molecules, particularly plant natural products (alkaloids, flavonoids, polyphenol and flavonoids, triterpenes and steroids) possessing potent HAase have been discovered. A number of plant species from various families, which have folk medicinal claims for these ailments (related to hyaluronan disturbances) were scientifically proven for their potential to block HAase enzymes.

**Keywords:** hyaluronidase inhibitors, natural products, medicinal plant, phytochemicals

#### **1. Introduction**

Hyaluronan/hyaluronic acid (HA) is a biologically important polysaccharide molecule found in the animal kingdom, most notably in the extracellular matrix (ECM) of connective tissues and on the surface of certain pathogenic bacteria. Although HA is found in nearly every tissue of vertebrates, it is abundantly present in the extracellular matrix of soft connective tissues. In mammals, it's predominantly found in the connective tissue of skin, testes, umbilical cord and synovial fluid. HA is composed of a linear polymeric chain with a uniform repeating disaccharide units of D-glucuronic acid and N-acetyl-D-glucosamine linked through (1,3 and 1,4) glycosidic bond. HA is a megaDalton molecule, synthesized as free polymer by the plasma membrane at its inner face [1–3].

The molecular function of hyaluronan in the body include interaction with HA receptors on the surface of the same cell or ECM molecules of the surrounding cells [4, 5]. When newly secreted, HA interacts with a variety of cell surface receptors

(CSRs) which give rise to important physiological functions such as signal transduction, building of pericellular matrix and the degradation endocytosis of HA via receptor-mediated internalization [6–8].

The metabolism of hyaluronan involves hyaluronidase enzyme, which is a class of glycosidase that predominantly degrades hyaluronan (HA). Karl Meyer coined the word Hyaluronidases (HAases), and over the years of research, the importance of HAases in controlling the physiological and pathological function of HA in animals has been established [9]. In mammals, the HAases hydrolyze the glucosaminidic β-1,4-linkages of hyaluronic acid and produces tetrasaccharide fragments. Three types of HAase enzymes act in concert to degrade HA biochemically; first, intact HA is acted on by endoglycosidase HAases, resulting in oligosaccharides with varying chain lengths that serve as substrates for the other two HAase enzymes (exoglycosidases), namely -glucuronidase and -N-acetyl hexos [10, 11].

The enzyme hyaluronidase and its substrate (Hyaluronan) perform a critical biological function in human body and their imbalance has been linked with various pathological processes and disease states including skin diseases and cancer [1]. The biological role of HA depends on the type of product formed after degradation and the circumstances under which it is synthesized [6, 12]. The involvement of HA has been established in various physiological and pathological processes include embryogenesis [7, 13, 14], immune surveillance, inflammation [15–18], wound healing [19], multi-drug resistance [6], cancer, water homeostasis and viscoelasticity of ECM [6, 8, 11, 20, 21]. Thus, it is critical to maintain HA homeostasis by balancing the action of HAase enzymes involved in anabolic and catabolic activities using various approaches such as hyaluronidase enzyme inhibitors (HAIs). The biological and therapeutic potential of HAase inhibitors (HAIs) is receiving significant attention, and an increasing amount of research is being conducted to develop potent hyaluronidase inhibitors for a variety of health conditions, including contraceptives, anti-tumor, antimicrobial, and anti-venom/toxin agents [22–24]. Hyaluronidase inhibitors of different chemical types are increasingly being reported, which include synthetic and plant derived bioactive compounds, polysaccharides, fatty acids, proteins, glycosaminoglycans and others [23, 25–30].

In this chapter, we have discussed and presented an updated overview of studies on important natural product agents (small molecules, and plants extracts) of various chemical forms derived from medicinal plants, which have been reported as potent hyaluronidase inhibitors. The search engines, such as, Google Scholar and PubMed were used to search the literature using key words such as natural products, medicinal plants, phytochemicals with hyaluronidase inhibitors, and antihyaluronidase. Majority of the data covered in this study are research published during the last fifteen years and studies with incomplete data or doubtful peer review system were excluded.

#### **2. Hyaluronidases**

Hyaluronidases are a family of endoglycosidase enzymes found in both eukaryotes and prokaryotes and prevalent across the animal kingdom [31]. It was first observed by Duran-Reynals in mammalian testis extract and termed it "spreading factor" as it has the property of breaking down the hyaluronan structure and facilitating tissue permeability and spreading [32]. Karl Meyer later classified hyaluronidases into three groups depending on chemical analysis and end products formed, which included mammalian, leech, and bacterial hyaluronidases.

Mammalian hyaluronidases are endo-β-Nacetlyhexosaminidases which arbitrary cleave hyaluronan glycosidic ate β-1-4 position, yielding even numbered tetra- and

*Plant Natural Products: A Promising Source of Hyaluronidase Enzyme Inhibitors DOI: http://dx.doi.org/10.5772/intechopen.98814*

hexa oligosaccharides as the major end products along with N-acetylglucosamine at the reducing end of the product. These hyaluronidases exhibit both hydrolytic and transglycosidase activity and are found in spermatozoa, mammalian cell lysosomes, and bee, snake, and reptile venoms [33].

The second type of HAases are leech hyaluronidase, which cleave glucoronate linkages of hyaluronan and are inert towards other glycosaminoglycans. These group of HAases are hyaluronate-3-glycanohydrolases are endo-β-Dglucuronidases. Tetra- and hexasaccharides are the main end products with glucuronic acid at the reducing end. This group of enzymes are present in salivary glands of leeches and hook worms [34].

The third type is microbial hyaluronidases, which are distinguished from mammalian and leech HAases by their lack of hydrolysis activity. These HAases catalyze the cleavage of HA at the 1–4 glycosidic bond, resulting in the formation of 4 and 5 member unsaturated oligosaccharides. Enzymes in this class includes HA lyases from *Streptococcus pneumoniae* (S. PHL) and *S. agalactiae* [34, 35].

In humans, six hyaluronidase-like genes known as hyaluronoglucosaminidases (Hyals1–6) have been identified. Of the six Hyal genes, Hyal1 and 2 are the primary hyaluronidases responsible for the catabolism of HA in somatic tissue, while Hyals3 to 6 are inactive and likely do not participate in HA cleavage [36]. Although inactive, hyal3 is widely expressed in chondrocytes, testis, and bone marrow, and its expression increases when fibroblasts differentiate into chondrocytes. Inflammatory cytokines such as IL-1 and TNF- (tumor necrosis factor-alpha) upregulate the Hyal2 and Hyal3 genes, but not the Hyal1 gene [37].

#### **3. Plant derived natural products as hyaluronidase inhibitors**

In the regulation of biological processes, inhibition of enzyme activity can be as essential as the activity itself. Many diseases are caused by overactivation of enzymes, which can be regulated with enzyme inhibitors since blocking the enzyme is more efficient in active catabolic reactions than stimulating the synthesis of substrates such as the high molecular weight polymeric hyaluronan contained in the extracellular matrix [38]. This is particularly true when a rapid response or finely regulated temporal and spatial ECM activities are required. Mio and his colleagues have identified the first inhibitor of the hyaluronidase enzyme in human and mouse serum [39].

For centuries, nature has been a source of medicinal products, with numerous useful medicines have been derived from plant sources [40]. Their therapeutic utility in treating a variety of illnesses have been investigated in various conventional medical systems, and their role as a biological modulator has been recognized throughout human history [41]. Natural products' effectiveness as enzyme inhibitors is attributed to their product biosynthetically in living organisms, which enhances their chances of interacting effectively with a variety of biological targets [42]. The inherent steric complexity, more number of rings and chiral centers, as well as the presence of more oxygen and the ability to form more hydrogen bonds, increases drug-likeness property of natural products from synthetic ones [43, 44]. The following section discusses recent research on various plant extracts and phytoconstituents as potential sources of hyaluronidase inhibitors.

#### **3.1 Anti-hyaluronidase phytoconstituents**

Various class of natural products derived from different plants species documented as hyaluronidase inhibitors include alkaloids, flavonoids, polyphenols, terpenes and steroids as shown in the **Table 1**. Natural products derived from plants



*Plant Natural Products: A Promising Source of Hyaluronidase Enzyme Inhibitors DOI: http://dx.doi.org/10.5772/intechopen.98814*

#### **Table 1.**

*Natural product compounds active against hyaluronidase enzyme.*

are well-known as HAase inhibitors due to their unique structural features. As indicated in **Table 1**, many classes of natural compounds produced from various plant species have been recorded as hyaluronidase inhibitors. These classes include alkaloids, flavonoids, polyphenols, terpenes, and steroids.

#### *3.1.1 Alkaloids*

Alkaloids are naturally occurring secondary metabolites, which consist of a basic nitrogen atom and produced by various species of animals, plants, bacteria and fungi. Morikawa and his team evaluated aporphine and benzylisoquinoline alkaloids which they have earlier isolated from the flower buds of Sacred lotus (*Nelumbo nucifera*) tably, Among the alkaloids discovered as a hyaluronidase inhibitor, nornuciferine (IC50 = 22.5 μM), asimilobine (11.7 μM), norarmepavine (26.4 μM), coclaurine (11.4 μM), and norjuziphine (24.3 μM) have shown potent activity, even higher than the standard atilllergic drug (disodium cromoglycate (IC50 = 64.8 μM). Nuciferine, N-methylasimilobine, pronuciferine, armepavine, and N-methylcoclaurine are the other alkaloids with moderate anti-HAase action [45]. Girish and co-researchers tested various compounds including well known alkaloids such as aristolocic acid, reserpine, and ajmaline on hyaluronidase enzymes obtained from the *Naja naja* snake venome and observed a dose dependent inhibition of hyaluronidase enzyme activity in manner. It was further observed that aristolochic acid has completely inhibited HAase, while reserpine and ajmaline inhibited it partially in a non-competitive manner. [28, 46]. Olgen and colleagues have tested as a series of aminomethyl indole alkaloids derivatives against the bovine testes hyaluronidase and found 3-[(4-methylpiperazin-1-yl)methyl]-5-phenyl-1H-indole as the most potent inhibitor of HAase enzyme [47].

#### *3.1.2 Flavonoids and polyphenols*

Flavonoids are a large group of polyphenolic compounds having benzo-γ-pyrone structure and are ubiquitously present in various parts of the plants. Flavonoids are a wide class of polyphenolic chemicals with a benzo—pyrone structure that are found in virtually every part of plants. Secondary metabolites of phenolic origin, such as flavonoids, are involved in a variety of pharmacological activities [28]. Based on their structure, flavonoids of different types such as flavones, anthocyanidines, flavones, and chalcones have demonstrated antioxidant, anti-inflammatory, antiviral, and antithrombotic properties, antitumor, hepatoprotective and enzyme inhibitory properties [48–50].

Girish and co-researchers have observed in an *in vitro* study that flavonoids of different structure types such as flavone, tannic acid quercetin were able to inhibit the hyaluronidase enzyme activity obtained from *Naja naja* snake venom [28].

In an early study, Rodney and co-researchers evaluated the effect of flavonoids on hyaluronidase and afterwards the effect of 31 flavonoids has been found potent against the activity of bovine testicular hyaluronidase. The inhibitory action of flavonoids on hyaluronidases is dependent on the number of hydroxyl groups and side chain substituents present in the molecules, and flavonoids containing many hydroxyl groups were found to significantly reduce inhibitory activity hyaluronidase enzyme [51]. Plant based flavonoids such as flavones, 2-hydroxy-flavone, apigenin, luteolin, quercetin, and myricetin demonstrated the inhibitory effects on hyaluronidase activity [51].

Herte et al. investigated the effects of several flavonoids on the microbial origin of the hyaluronidase enzyme (Hyaluronate lyases). During their research, they discovered quercetin and myricetin to be the most potent inhibitors, with extra hydroxyl groups at positions 3,3′ (quercetin) and 5′ (myricetin) (myricetin). In addition, glycosylated flavonoids such as rutin, apiin, and silybin have shown a decline in their capacity to inhibit hyaluronate lyase, even when the side groups carried hydroxyl groups themselves [52].

*Plant Natural Products: A Promising Source of Hyaluronidase Enzyme Inhibitors DOI: http://dx.doi.org/10.5772/intechopen.98814*

A series of flavonoids were examined by Kuppusamy et al. against hyaluronidase enzyme extracted from the venom of honey bee, scorpion and cobra and found flavonoids such as myricetin, quercetin, luteolin, apigenin, phloretin and kaempferol showing potent anti-HAase effects in *in vitro* assay [53]. In another study the same authors observed a contrast where, sylibin inhibited hyaluronidase activity of bee and scorpion venom supported by the apigenin, kaempferol, luteolin, and tannic acid [54].

Kim and his co-worker isolated flavonols (quercetin 3-O-β-D-glucopyranoside, quercetin 3-O-β-D-xylopyranoside, kaempferol 3-O-β-D-glucopyranoside, and isorhamnetin 3-O-β-D-glucopyranoside) from the *Allium sativum* L. for antihyaluronidase properties [55].

Polyhenols are naturally occurring secondary metabolites, largely found in plants and generally involves in the defense of plants against pathogens [56]. Other type of phenolic compounds includes rosmarinic acid, lithospermic acid B, diometin-7-Oβ-D-glucopyraanoside, and apigenin-7-O-β-D-glucuronopyranoside reported from the *Meehania fargesii* plant, all of which were effective at suppress HAase activity [57]. Tatemoto and colleagues investigated the effects of tannic acid, apigenin, and quercetin, in *in vitro* fertilization parameters, on hyaluronidase activity, and found that tannic acid was the most active hyaluronidase enzyme activity in a dosedependent manner at concentrations ranging from 2 to 10 g/ml [58]. The same group of researchers investigated three tannins, tannic acid, gallic acid, and ellagic acid, and found tannic and ellagic acid as potent inhibitors of the hyaluronidase enzyme, effectively preventing polyspermy by suppressing the acrosome reaction induced by sperm-zona interaction during in vitro fertilization of porcine oocytes [59].

#### *3.1.3 Terpenes and steroids*

Terpenes are the constituents of pheromones, anti-feedants and flavors, which are composed of isoprene unite (C5) and their derivatives. Terpenes and terpenoids (oxygenated derivative) are recognized as one of the important class of natural products, are widely distributed in plants and possesses a range of bioactivity, exhibiting a wide bioactivity, such as anticancer, neuroprotection, and anti-inflammation and anti-infective agents [60, 61]. Abdullah and co-authors isolated teriterpenes as HAase blocking agents from *Prismatomeris tetrandra* (Roxb.) K. Schum. The two triterpenoids (3β-urs-12-en-28-oic acid and 3β,19,23-trihydroxyurs-12-en-28-oic acid) were obtained from the chloroform fraction whereas another triterpenoid 3β-acetylolean-12-en-28-oic acid was isolated from the roots of *Prismatomeris tetrandra*. Also, the synthetic analogues of ursolic acid were identified as potential inhibitor of hyaluronidase [62]. The *in-vitro* inhibition of bovine hyaluronidase and hylaluronate lyase was shown by the triterpenes glycyrrhizin and glycyrhetinic acid [52]. However, fatty acid derivative of glycyrrhetinic acid, known as stearyl ester was unable to inhibit the hyaluronidase activity [63]. This difference may be due to the specific structures of the respective enzymes as well as the splitting mechanism of hyaluronic acid as endoenzyme or exoenzyme [64].

Sterols are important structural components in higher organisms. They take part in the regulation of membrane fluidity, permeability and membrane associated metabolic processes [65]. Steroids of different structure types are reported to influence hyaluronidase metabolism [66]. Patil and co-researchers found the steroidal fraction isolated from the leave of *Carissa carandas* as strong inhibitior of hyaluronidase enzyme activity with IC50 = 5.19 mM/mL as compared to the standard (quercetin). This steroidal fraction could contain potential hyaluronidase inhibitor and therefore should be considered for further studied as anti-venom agent [67].

In a study, Lengers and team found chicoric acid (IC50 = 171 μM) and testosterone propionate (IC50 = 124 ± 1.1 μM) as strong inhibitors of Hyal1 expressed over the surface of *Escherichia coli* F470 which was comparable to that of glycyrrhizic acid (IC50 = 177 μM) [68].

#### **3.2 Anti-hyaluronidase medicinal plant extracts**

Plants have remained a major source of medicine for centuries and therapeutic agents derived from natural sources are used traditionally to recover from wound healing, treat snakebites or inflammation as contraceptives. Several studies indicate that plants species from various families, which have folk medicinal claims for these ailments were also scientifically been proven for their potential to block HAase enzymes as shown in **Table 2**.



*Plant Natural Products: A Promising Source of Hyaluronidase Enzyme Inhibitors DOI: http://dx.doi.org/10.5772/intechopen.98814*


#### **Table 2.**

*Medicinal plants with hyaluronidase activity.*

In a bioassay directed study, the polar fraction of *Aesculus hippocastanum* L (seeds) and *Hedera helix* L (leaves) were found active against hyaluronidase enzyme and later isolation of triterpene and steroidal saponins and sapogenins also exhibited strong anti-HAase activity when tested against testicular hyaluronidase enzyme [69].

The well-known medicinal plant *Hygrophila schulli*, traditional used as antiinflammatory and pain treatment in the north Ethiopia and India, possess antihyaluronidase activity *in vivo* by the ethanolic leaf extract [70]. In a study by Lee et al. [71], the aqueous methanolic extracts of 150 plant species assayed for their potential as hyaluronidase inhibitors, the extracts of six species found to be most active against HAases were *Areca catechu*, *Alpinia katsumadai*, *Dryopteris cassirrhizoma*, *Cinnamonum cassia*, and *Curcuma longa*, and the extract of *Areca catechu* showed relatively higher anti-HAase activity. The major constituents identified in *Areca catechu* which include phenolic compounds such as flavonoids and tannins could be responsible for the anti-HAase effect [72].

In another anti-HAase screening study, Tomohara et al. [73] evaluated the decoction extracts of 98 plant species for HAase inhibitory activity in an *in vitro* HAase assay. They observed 17 extracts exhibited moderate to high inhibitory activity (>50% inhibition at 500 μg/mL) and also noted correlation between the total phenol present in the extract and their cumulative effect as anti-HAase activity was varying. From the study, rhizome extract of *Panax japonicus* and root bark extract of *Prunus salicina* were found most potent HAase inhibitors.

Jeong et al. [74] evaluated 100 Korean medicinal plants for their anti-allergic activity. The methanolic rhizome extract of *Anemarrhena asphodeloides* was found active against hyaluronidase enzyme. Marquina and colleagues investigated the anti-inflammatory effect of blackberry fruit extract and fractions. Two of the seven fractions inhibited the hyaluronidase enzyme and shown superior anti-inflammatory effect when compared to aspirin [75].

A study conducted by Liyanaarachchi et al. [76] on fifteen Sri Lankan medicinal plants for their skin aging and anti-wrinkle effect has identified three plant extract with relatively higher anti-HAase activity. The ethanol extract of *Curcuma* 

#### *Plant Natural Products: A Promising Source of Hyaluronidase Enzyme Inhibitors DOI: http://dx.doi.org/10.5772/intechopen.98814*

*aromatica* rhizomes exhibited marked hyaluronidase inhibitory activities (95.0%) inhibition at 500 μg/mL) followed by *Artocarpus altilis* (68.59%) and *Artocarpus nobilis* bark extracts (44.78%) when tested at 500 μg/mL concentration level.

Selenge et al. [77] studied two medicinal plant famous in Mongolian traditional medicine *Chamaerhodos erecta* and *C. altaica* and revealed in the bioassay guided isolation the moderate ability of its constituents as anti-HAase enzyme, suggesting their potential to prevent the extracellular matrix degradation factors.

Similarly, the Sri Lankan origin black tea *Camellia sinensis* L. (Orthodox Orange Pekoe) was evaluated for its potential as cosmeceutical for skin aging by Ratnasooriya et al. [78]. The extract revealed moderate anti-HAase activity (IC50 =1.09 ±0.12 mg/mL) compared to standard compound (epigallocatechin gallate) (IC50 = 0.09±0.00 mg/mL) in as dose dependent manner.

Han et al. [79] assayed the fermented and non-fermented seed's methanolic seed extract of White Sword Beans (*Canavalia gladiata* DC) and Soybeans (*Glycine max* L. Merrill) and found higher inhibitory activity exhibited by red sword beans (nonfermented/fermented) (1.5–2.6-fold) against HAase enzyme than that of soybeans (non-fermented/fermented). The study suggests that *B. subtilis*-fermented sword beans are potential as potential anti-inflammatory agents for the food industry.

Furusawa et al. [80] investigated the silverskin coffee beans (a by-product during roasting) for its anti-inflammatory and anti-allergic effects. The results indicated a potent inhibitory effect against hyaluronidase (IC50=0.27 ±0.04 mg/mL) as compared to the standard disodium cromoglycate (IC50 = 0.31±0.05 mg/mL). The strong effect is argued possibly due to the presence of acidic polysaccharides present in the extract, which is mainly composed of uronic acid present in Silverskin coffee beans extract.

A major screening study on 500 Korean Medicinal plants as HAase inhibitors identified the stem extract of three species possessing relatively higher anti-HAase activity include plant specied *Styrax japonica* (57.28%), *Deutzia coreana* (53.50%), and *Osmanthus insularis* (53.19%). The study further explores that the HAase inhibition could be due to presence of multifunctional compounds and may be effective in preventing allergic reactions and inflammation [81].

*Dracocephalum foetidum* Bunge, is a medicinal plant traditionally used by Mongolian nomads for various infections and suppurative disease and fever. Its chemical and physiological role was investigated by Selenge et al. [82] and found the aqueous acetone extract of the aerial part possess potent anti-hyaluronidase activity Acetone extract (IC50 = 0.27 ± 0.01 mg/mL) compared to the standard (disodium cromoglicate, IC50 = 0.33 ± 0.02 mg/mL) and further phytochemical analysis of its aqueous fraction resulted into compounds of various class as highly potent HAase inhibitors.

Załuskia et al. [83] found strongest inhibitory effects in the freshly dried fruits of *Eleutherococcus senticosus* (IC50 = 0.58 ± 0.01 mg/mL) and *E. henryi* (IC50 = 0.61 ± 0.05 mg/mL), compared to positive control (Methyl indole-3-carboxylate, (IC50 = 07.11 mM). *Eleutherococcus senticosus* Maxim. called as Siberian ginseng, and has been used from ancient times in Northeastern Asia and Eastern Russia as a tonic and anti-fatigue agent. In northeast China, the ethanolic roots extract is a popular health supplement for weakness, rheumatism, impotence and hemorrhoids [84, 85]. *E. senticosus* products are imported in Europe and it is one of the ten popular herbal dietary supplements in North America [86].

Murata and co-researchers investigated three the 80% acetone extracts of three medicinal plants, *Keiskea japonica, Lycopus lucidusand* and *Meehania fargesii* for their inhibitory effect against hyaluronidase. From bioassay directed study of these plants, the phenylpropanoids and flavone glucuronide from aerial part of *Keiskea japonica* [87], phenylpropanoids from aerial parts *Lycopus lucidas* [88] and spermidine alkaloids flavone glycosides from dried whole plant extract of *Meehania fargesii* [57] showed strong hyaluronidase inhibitory activity.

Piwowarski and group examined tannin-rich aqeous extract of twelve plant for their ability to inhibit hyaluronidase materials based on their use in traditional Polish medicine for external treatment of skin and mucosal diseases. Among the plants, *Lythrum salicaria* L. extract has shown strongest inhibition of hyaluronidase (IC50 = 8.1 ± 0.8 μg/mL) compared to the heparin (IC50 = 62.1 ± 7.5 μg/mL) which was used as standard control [89].

*Terminalia chebula*, an Indian medicinal plant was assessed for its role as male antifertility agent using hyaluronidase inhibition enzyme assay. The 95% ethanol dried fruit extract of the plant showed in vitro HAase inhibitory activity of the human spermatozoa (~93% inhibition,) (IC50 = 0.8579 mg/ml) and rat caudal epididymal spermatozoa (~86% inhibition) (IC50 = 1.6221 mg/ml) at 30 mg/mL compared to the standard tannic acid (IC50 =299.6 μM). In the *in vivo* study on rates showed statistically significant (P < 0.001) inhibition of hyaluronidase activity of HAase enzyme extracted from testis (50 mg/kg dose, −47% decrease) and caused a further decrease (−72% decrease) at 100 mg/kg dose. The anti-HAase activity of the extract against caput and cauda epididymal spermatozoa extracted enzyme exhibited significantly better (P < 0.001) activity at 50 mg/kg dose (−41% each) and 100 mg/kg dose (−65% and −77%, respectively) when given orally for 60 days [90].

Michel and colleagues investigated the anti-inflammatory properties of Eastern Theaberry (*Gaultheria procumbens*) [91], found the chloroform (IC50 = 282.15 ± 10.38 μg/mL) and pet-ether (IC50 = 401.82 ± 16.12 μg/mL) fractions of the plant leaf extract as potent hyaluronidase inhibitors compared to the standard drug heparin (IC50 = 366.24 ± 14.72 μg/mL) which was higher than the activity they observed in nine most active constituents present in the sample.

Citalingam and Co-researchers have screened different extracts prepared from the bark and leaves of *Payena dasyphylla* medicinal plants for their potential as antihyaluronidase inhibitors. It was found to exert higher activity (IC50 = 100 μg/mL) against bovine testicular hyaluronidase The *Payena dasyphylla* extract also showed strong inhibition of HAase expression (IC50 = 100 ng/mL) in the cultured human chondrocyte cells in response to IL-1β. Similarly, the ethyl acetate fraction of the plant has strongly exhibited inhibited the HYAL1 and HYAL2 mRNA gene expressions (IC50 = 100 μg/mL) [92].

*Phyllanthus emblica* is a rejuvenating plant famous in Ayurvedic medicine has been evaluated by Sumantran et al. for its ant-arthritic property [93]. The aqueous decoction extract fruit (powder) was found to inhibit the activity of HAase enzyme effectively at IC50 = 0.15 mg/ml.

The muscadine grape (*Vitis rotundifolia*) is scientifically known for its antiinflammatory properties. Bralley and co-authors have tested the ethanol extract of fruit skin and seed for their inhibitory potential against hyaluronidase enzyme. In their study they observed the bronz IC50 = 0.3 mg/mL for) and purple IC50 = 0.6, mg/mL) mascadine seed extracts as highly potent compared to the fruit skin extracts of the two mascadine types (IC50 = 1.0, 1.0 mg/mL respectively) [94].

*Malaxis acuminata*, an important medicinal plant known in Ayurvedic medicine was evaluated for its effect on skin aging and related enzyme activity. The researchers found the in vitro- isolated leaf extract (Methanolic) as strong inhibitor of hyaluronidase activity (IC50 = 60.36 ± 1.6 μg/mL) compared to the standard compound oleanolic acid-known for skin protective effect (IC50 = 32.45 ± 1.7 μg/mL) [95].

Girish et al. demonstrated that the aqueous root extract of *Mimosa pudica* reduced the hyaluronidase activity of three Indian snake venoms; *Naja naja* (2.16 x 10−3 ± 0.04), *Vipera russelii* (1.25 x10−3 ± 0.045) and *Echis carinatus* (1.1 x10−3 ± 0.072) units/min/mg protein [96].

*Plant Natural Products: A Promising Source of Hyaluronidase Enzyme Inhibitors DOI: http://dx.doi.org/10.5772/intechopen.98814*

Plants bearing high amount of tannin are known to block Hyaluronidase enzyme. A study conducted by Granica et al. discovered the extracts made from aerial part of two plants Oenothera paradoxa Hudziok and *O. biennis* L, which are rich in macrocyclic ellagitannin showed strong inhibition (97.3 ± 3.0% and 97.9 ± 1.7% respectively) of the HAase enzyme activity at 50 μg/mL compared to the stander heparine (62.1 ± 7.5 μg/mL) [97].

In a recent study on *Otostegia fruticose*, a medicinal plant traditionally used in Ethiopia to treat different ailments including inflammatory disorders. In the study, Bahta and co-researchers [98] found the ethanolic leaf extract and its fractions a dose depended anti-HAase activity. The crude ethanolic extract and chloroform fraction exhibited highest hyaluronidase inhibition (79.20% and 85.75% respectively), compared to standard drug indomethacin (95.52%) at the concentration of 100 μg/mL.

Brown algae are a nutrient-dense and potential source of bioactive secondary metabolites. In a study conducted by Shibata and co-workers [99], the crude phlorotaninin extract of two brown algae (*Eisenia bicyclis* and *E. kurome*) exhibited stronger anti-HAase activity (IC50 = 0.03 and 0.035 mg/mL respectively) compared to the two standard compounds Epigallocatechin gallate (IC50 = 190 mM/mL and Sodium cromoglycate (IC50 = 270 mM/mL). In the same study they observed its constituents possessing strong inhibitory activity.

In another study on algae, Fayad and co-researcher have used capillary electrophoresis-based enzymatic assay method to assess the anti-skin aging property of a macroalga (*Padina pavonica*). In their study, the water extract was found strongly inhibiting the hyaluronidase activity (IC50 = 0.04 ± 0.01 mg/mL) compared to the literature reported value of phlorotannin fractions of *Einsenia bicyclis* (IC50 = 0.03 mg/mL) [100].

#### **4. Conclusion**

The modulation of hyaluronidase enzyme and its substrate HA throughout the body is critical to maintain hyaluronan homeostasis as HA degradation is associated with pathogenesis of various health conditions. The literature survey carried out in this study found an increasing number of studies reported on HAase inhibitors derived from various biological sources and majority of the discoveries were from medicinal plants which have ethnobotanical claims for ailments associated with hyaluronan. Various class of natural products identified include alkaloids, flavonoids and terpenes have shown potent inhibitory activity against HAases in the *in vitro* studies. Similarly, a number of medicinal plant extracts and their fractions were found active against hyaluronidases and could serve as potential reservoirs for HAase inhibitors. These preliminary findings need further research to identify the active constituent(s) present in the extracts and establish their mechanism of action, safety profile and appropriate dosage of the active agents in animal and human studies. Hence, HAase inhibitors could be effective in controlling diseases involving uncontrolled degradation of HA and may serve as chondroprotective, anti-aging, antitumor, antimicrobial contraceptive agents, and anti-venom alternative.

#### **Acknowledgements**

The authors are thankful to the Department of Biological Sciences and NUMS Institute for Advanced Studies and Research (NIASR), National University of

Medical Sciences, Rawalpindi, Pakistan for supporting this study. We also apologize to the authors of many interesting studies that were omitted due to limitation.

### **Conflict of interest**

The authors declare that they have no conflict of interest.

### **Author details**

Muhammad Zeeshan Bhatti and Aman Karim\* Department of Biological Sciences, National University of Medical Sciences, Rawalpindi, Pakistan

\*Address all correspondence to: aman.karim@numspak.edu.pk

© 2021 The Author(s). Licensee IntechOpen. This chapter is distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/ by/3.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.

*Plant Natural Products: A Promising Source of Hyaluronidase Enzyme Inhibitors DOI: http://dx.doi.org/10.5772/intechopen.98814*

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