Natural product compounds active against hyaluronidase enzyme.
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.
- hyaluronidase inhibitors
- natural products
- medicinal plant
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, 2, 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, 7, 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 . 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 . 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, 16, 17, 18], wound healing , multi-drug resistance , 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, 23, 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, 26, 27, 28, 29, 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 anti-hyaluronidase. 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.
Hyaluronidases are a family of endoglycosidase enzymes found in both eukaryotes and prokaryotes and prevalent across the animal kingdom . 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 . 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 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 .
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-D-glucuronidases. 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 .
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
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 . 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 .
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 . 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 .
For centuries, nature has been a source of medicinal products, with numerous useful medicines have been derived from plant sources . 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 . 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 . 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 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.
|Class of Natural Products||Compounds||Source of HAase enzyme||IC50/%Inhibition||Ref.|
|Aristolochic acid||50 μM|||
|Aristolocic acid||Naja naja venom||1.43 μM|||
|3-[(4-methylpiperazin-1-yl)methyl]-5-phenyl-1H-indole||Testicular||23% (5 μM)|||
|Naja naja venom||50 μM|||
|Tannin||Honey bee, scorpion, snakes and cobra venoms||0.9%|||
|Honey bee, scorpion, snakes and cobra venoms|||
|quercetin 3-O-β-D-glucopyranoside||Bovine testes||20.9 mM|||
|quercetin 3-O-β-D-xylopyranoside||22.1 mM|
|kaempferol 3-O-β-D-glucopyranoside||26.5 mM|
|Rosmarinic acid||Testicular||309 μg/mL|||
|Lithospermic acid B||164 μg/mL|
|Apigenin-7- O-β-D-glucuronopyranoside||548 μg/mL|
|Tannic acid||Testicular||4.97 units/mL|||
|Tannic acid||Testicular||0.8 units/mL|||
|Gallic acid||5 units/mL|
|Ellagic Acid||4.8 units/mL|
|Chicoric acid||171 μM|||
|Glycyrrhetinic acid||Bovine testes||0.060–0.260 mM|
|3β-urs-12-en-28-oic acid||Testicular||103.18 ±1.70 μM|||
|3β,19,23-trihydroxyurs-12-en-28-oic acid||286.95±10.28 μM|
|3β-acetylolean-12-en-28-oic acid triterpenoid||1466.5± 2.37 μM|
|Steroidal Fraction||5.19 mM|||
|Testosterone Propionate||124 ± 1.1 μM|||
|Glycyrrhizic acid||175 ± 1.2 μM|||
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 (
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 . 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, 49, 50].
Girish and co-researchers have observed in an
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 . Plant based flavonoids such as flavones, 2-hydroxy-flavone, apigenin, luteolin, quercetin, and myricetin demonstrated the inhibitory effects on hyaluronidase activity .
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 .
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
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
Polyhenols are naturally occurring secondary metabolites, largely found in plants and generally involves in the defense of plants against pathogens . Other type of phenolic compounds includes rosmarinic acid, lithospermic acid B, diometin-7-O-β-D-glucopyraanoside, and apigenin-7-O-β-D-glucuronopyranoside reported from the
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
Sterols are important structural components in higher organisms. They take part in the regulation of membrane fluidity, permeability and membrane associated metabolic processes . Steroids of different structure types are reported to influence hyaluronidase metabolism . Patil and co-researchers found the steroidal fraction isolated from the leave of
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 Name||Plant part/type of extract (active extract)||Biological activity||Source of HAase enzyme||Ref.|
|Whole plants 80% acetone extract (water soluble fractions)||Anti-HAase||Testicular|||
|Whole plant/aqueous methanol||Anti-aging||Testicular|||
|Methanol–water extract of whole plant||Anti-aging/Anti-inflammatory||Testicular|||
|Methanol–water extract of whole plant||Anti-aging/Anti-inflammatory||Testicular|||
|Root bark/aqueous decoction||Anti-HAase||—|||
|Aerial parts/80% acetone extract (aqueous fraction, BuOH)||Anti-HAase||—|||
|Leaves, buds/(water brew)||Anti-aging/skin care||Testicular|||
|Seeds/80% methanol extracts(fermented and non-fermented)||Anti-inflammatory||—|||
|Seeds/80% methanol extracts (fermented and non-fermented)||Anti-inflammatory||—|||
|Seeds/Coffee silverskin (byproduct of the roasting procedure for coffee beans||Anti-inflammatory/Anti-allergy||Testicular|||
|Aerial parts/80% aqueous acetone extract (aqueous fraction)||Anti-inflammatory||Testicular|||
|Aerial part/80% acetone extract||Anti-HAase||Testicular|||
|Aerial part 80% acetone extract||Anti-HAase||Testicular|||
|Whole Plant/aqueous extract||Anti-inflammatory||Testicular|||
|Fruit dried/95% ethanol extract||Anti-fertility||Human spermatozoa|||
|Leaves/Petroleum ether, chloroform||Anti-inflammatory||Testicular|||
|Aqueous extract of fruit||Chondroprotective||Testicular|||
|Seed and skin/50% ethanol extract||Anti-HAase||Testicular|||
|Leaves, stem/methanolic extract||Skin-aging||—|||
|Roots/aqueous extract||Anti-ophidian||Snake venom|||
|Aerial Part/50% methanolic extract||Anti-inflammatory||—|||
|Aerial Part/50% methanolic extract||anti-inflammatory||—|||
|Leaf/70% ethanolic extract||Anti-inflammatory||Testicular|||
|Brown algae (||Crude phlorotaninin extract||Anti-aging||Testicular|||
|Padina pavonica (Dictyotaceae)||Seaweed/extracts (Pressurized liquid extraction, microwave assisted extraction. Supercritical fluid extraction)||Anti-aging||Testicular|||
In a bioassay directed study, the polar fraction of
The well-known medicinal plant
In another anti-HAase screening study, Tomohara et al.  evaluated the decoction extracts of 98 plant species for HAase inhibitory activity in an
Jeong et al.  evaluated 100 Korean medicinal plants for their anti-allergic activity. The methanolic rhizome extract of
A study conducted by Liyanaarachchi et al.  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
Selenge et al.  studied two medicinal plant famous in Mongolian traditional medicine
Similarly, the Sri Lankan origin black tea
Han et al.  assayed the fermented and non-fermented seed’s methanolic seed extract of White Sword Beans (
Furusawa et al.  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
Załuskia et al.  found strongest inhibitory effects in the freshly dried fruits of
Murata and co-researchers investigated three the 80% acetone extracts of three medicinal plants,
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,
Michel and colleagues investigated the anti-inflammatory properties of Eastern Theaberry (
Citalingam and Co-researchers have screened different extracts prepared from the bark and leaves of
The muscadine grape (
Girish et al. demonstrated that the aqueous root extract of
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
In a recent study on
Brown algae are a nutrient-dense and potential source of bioactive secondary metabolites. In a study conducted by Shibata and co-workers , the crude phlorotaninin extract of two brown algae (
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 (
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
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.