WO2024018437A1 - A new high molecular weight of hyaluronic acid or salt thereof of plant origin for use in maintaining joint homeostasis and preventing the harmful processes of osteoarthritis. - Google Patents

A new high molecular weight of hyaluronic acid or salt thereof of plant origin for use in maintaining joint homeostasis and preventing the harmful processes of osteoarthritis. Download PDF

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WO2024018437A1
WO2024018437A1 PCT/IB2023/057469 IB2023057469W WO2024018437A1 WO 2024018437 A1 WO2024018437 A1 WO 2024018437A1 IB 2023057469 W IB2023057469 W IB 2023057469W WO 2024018437 A1 WO2024018437 A1 WO 2024018437A1
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molecular weight
kda
hyaluronic acid
osteoarthritis
high molecular
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French (fr)
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Giorgio Stefano CERANA
Peter Bos
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VIVATIS PHARMA ITALIA S.r.l.
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    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K36/00—Medicinal preparations of undetermined constitution containing material from algae, lichens, fungi or plants, or derivatives thereof, e.g. traditional herbal medicines
    • A61K36/06—Fungi, e.g. yeasts
    • A61K36/07—Basidiomycota, e.g. Cryptococcus
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K36/00—Medicinal preparations of undetermined constitution containing material from algae, lichens, fungi or plants, or derivatives thereof, e.g. traditional herbal medicines
    • A61K36/06—Fungi, e.g. yeasts

Definitions

  • the present invention relates to new high molecular weight of hyaluronic acid or salt thereof (HMWHA) of plant origin (called Geenluronic®) for use in maintaining joint homeostasis and preventing the harmful processes of osteoarthritis. Further, the present invention relates to a process for production of said new high molecular weight of hyaluronic acid or salt thereof (HMWHA) of plant origin (called Geenluronic®).
  • HMWHA hyaluronic acid or salt thereof
  • the present invention refers to a composition
  • a composition comprising a mixture that comprises or, alternatively, consists of said new high molecular weight of hyaluronic acid or salt thereof (HM HA) of plant origin (called Geenluronic®), preferably said composition and mixture is for use (I) in maintaining joint homeostasis and preventing the harmful processes of osteoarthritis, and/or (ii) in a method for preventing and restoring the degradation of cartilage, and/or (ill) in a method for the treatment of osteoarthritis.
  • HM HA hyaluronic acid or salt thereof
  • Geenluronic® hyaluronic acid or salt thereof
  • Osteoarthritis is a slow progressive joint disorder which causes several disabilities in the adult population [1], For a long time, OA was regarded as a progressive wear of the joint cartilage alone. However, recent research has shown that it is an inflammatory disease of the entire synovial joint, which includes not only the mechanical degeneration of the articular cartilage but also the concomitant structural and functional change of the entire joint, including the synovium, meniscus, periarticular ligaments and subchondral bone [Loeser RF, Goldring SR, Scanzello OR, Goldring MB. Osteoarthritis: a disease of the joint as an organ. Arthritis Rheum. 2012 Jun;64(6): 1697-707. doi: 10.1002/art.34453.
  • OA OA
  • non-pharmacological e.g. physiotherapy
  • pharmacological e.g. steroidal and non-steroidal-anti-inflammatory drugs
  • intra-articular e.g. injection of hyaluronic acid
  • therapies can reverse the symptoms only in a small number of cases, but they don't stop the degeneration process of the cartilage or promote the repair of the tissue.
  • HA hyaluronic acid
  • GAGs glycosaminoglycan heteropolysaccharides family
  • Golgi enzymes Golgi enzymes
  • HMWHA high-molecular weight HA
  • Hyaluronic Acid Molecular Mechanisms and Therapeutic Trajectory. Front Vet Sci. 2019;6:192. Published 2019 Jun 25. doi: 10.3389/fvets.2019.00192], however, it can be suggested that the characteristic steric configurations HMWHA are needed for the manifestation of the analgesic effect indicating possible clinical applications of all fragments of HA [Gupta RC, Lail R, Srivastava A, Sinha A. Hyaluronic Acid: Molecular Mechanisms and Therapeutic Trajectory. Front Vet Sci. 2019;6: 192. Published 2019 Jun 25.
  • HA is one Food and Drug Administration (FDA) approved treatment for inflammatory conditions, including those affecting the joints, and is also acknowledged in Europe for its beneficial properties due to the therapeutic potential caused by native HA, but without toxicity [7].
  • FDA Food and Drug Administration
  • IL interleukin
  • PGE2 prostaglandin E2
  • HA may be combined with several agents including lactose-modified chitosan and cyclodextrin to improve chondroprotection and to stimulate cartilage growth reducing inflammation [Tarricone E, Elia R, Mattiuzzo E, et al. The Viability and An-ti-lnflammatory Effects of Hyaluronic Acid-Chitlac-Tracimolone Acetonide- p-Cyclodextrin Complex on Human Chondrocytes. Cartilage. 2021; 13(2_suppl):920S-924S.
  • HMWHA histoneum kinase phosphatase
  • HMWHA after the binding with CD44 is able to prevent chondrocytes apoptosis by inhibiting PGE2 synthesis and interleukin activity such as IL-1p which is responsible of oxidative stress [2, 17, 21 , 22],
  • IL-1p interleukin activity
  • the intermediate HA and HMWHA have antioxidant and antimicrobial properties [23]
  • This substance has the ability to regulate tissue homeostasis and its physiological decrease is related to the aging process that leads to various diseases [23]
  • Oral administration of exogenous HA has attracted the attention of researchers as a supplementary therapy to prevent or treat the aging process of cartilage and related diseases [Ewald CY (2021) Drug Screening Implicates Chondroitin Sulfate as a Potential Longevity Pill. Front. Aging 2:741843. doi: 10.3389/f rag i.2021.741843],
  • composition comprising a mixture that comprises or, alternatively, consists of said new high molecular weight of hyaluronic acid or salt thereof (HMWHA) of plant origin for use (i) in maintaining joint homeostasis and preventing the harmful processes of osteoarthritis, and/or (ii) in a method for preventing and restoring the degradation of cartilage, and/or (iii) in a method for the treatment of osteoarthritis OA.
  • HMWHA hyaluronic acid or salt thereof
  • the present invention relates to new high molecular weight of hyaluronic acid or salt thereof (HMWHA) of plant origin (called Geenluronic®) for use in maintaining joint homeostasis and preventing the harmful processes of osteoarthritis. Further, the present invention relates to a process for production said new high molecular weight of hyaluronic acid or salt thereof (HMWHA) of plant origin (called Geenluronic®).
  • the present invention refers to a composition
  • a composition comprising a mixture that comprises or, alternatively, consists of said new high molecular weight of hyaluronic acid or salt thereof (HMWHA) of plant origin (called Geenluronic®), preferably said composition and mixture is for use (i) in maintaining joint homeostasis and preventing the harmful processes of osteoarthritis, and/or (ii) in a method for preventing and restoring the degradation of cartilage, and/or (iii) in a method for the treatment of osteoarthritis.
  • HMWHA hyaluronic acid or salt thereof
  • Geenluronic® hyaluronic acid or salt thereof
  • the present invention refers to a mixture that comprises or, alternatively, consists of:
  • HMWHA high molecular weight of hyaluronic acid or salt thereof (HMWHA) of plant origin present that comprises or, alternatively, consist of disaccharide ADi-HA obtained by chondroitinase AC enzymatic hydrolysis of the Tremella extract.
  • the high molecular weight of hyaluronic acid or salt thereof (HMWHA) of plant origin does not contain chondroitin 4 and 6 mono-sulfates.
  • the high molecular weight of hyaluronic acid or salt thereof (HMWHA) of plant origin has a content of glucuronic acid comprised in a range from 80% to 98%, preferably from 85% to 95%, for example 90%.
  • the high molecular weight of hyaluronic acid or salt thereof (HMWHA) of plant origin has an average molecular weight comprised in a range from about 1500 kDa to about 20000 kDa, preferably from about 1650 kDa to about 15000kDa, more preferably from about 2000 kDa to about 10000 kDa, for example about 3000 kDa, about 4000 kDa, about 5000 kDa, about 6000 kDa, about 7000 kDa, about 8000 kDa, or about 9000 kDa.
  • said high molecular weight of hyaluronic acid or salt thereof (HMWHA) of plant origin has a higher amount of HA that crosses the barrier and reaches the plasma level compared to control (p ⁇ 0.0001 ) and compared to sodium hyaluronate (about 30%, p ⁇ 0.0001) with the greatest effects between 4h and 5h.
  • said high molecular weight of hyaluronic acid or salt thereof (HMWHA) of plant origin in an amount of 1 pig/pil is was able to reduce the negative effect produced by LPS (about 1.25 times more) better than sodium hyaluronate (about 50%, p ⁇ 0.05).
  • said mixture is for use:
  • the present invention also refers to a composition
  • a composition comprising said mixture and, optionally, at least one excipients or carrier of pharma or food grade, wherein said composition is for use:
  • said composition is preferably a food or nutraceutical composition, more preferably for oral use.
  • the present invention also refers to a process for preparing said mixture, wherein said process comprises steps of extraction of the high molecular weight of hyaluronic acid or salt thereof (HMWHA) from White Tremella (Silver Ear), purification, refining by alcohol solution, sieving and crushing to provide a powder of HMWHA.
  • HMWHA high molecular weight of hyaluronic acid or salt thereof
  • Glycosaminoglycans are linear polysaccharides formed from covalently bound disaccharide units. Their disaccharide repeating unit consists of an amino sugar (exoamines, including d-glucosamine and d-galactosamine) and a uronic acid (D-glucuronic acid and l-hyduronic acid). GAGs are generally covalently bound to a basic protein to form a proteoglycan with different physiological functions. They differ in chain length, protein binding, degree of sulphation and proportion of uronic acids, among others.
  • HA is a polysaccharide formed from long, repeated disaccharide units comprising N-acetyl-d-glucosamine (GalNAc) and d-glucuronic acid (GlcA) and is the only GAG that is not sulphated and not bound to proteins
  • CS is a GAG formed from repeated disaccharides of GalNAc and GlcA with a shorter chain than HA (20-100 repeated units) with a structure that differs according to the different positions of the sulphate groups [https: //doi.
  • GAGs differ in the amount of HA and CS recovered through the use of specific enzymes and/or solvents and also in the source of biomass used [https://doi.Org/10.1016/j.carbpol.2020.116441].
  • TFPS Tremella fuciformis polysaccharide
  • the monosaccharides detected in TFPS include mannose, xylose, fucose, glucose, galactose and, in particular, glucuronic acid.
  • TFPS is a mannitan formed by an a -(1 ,3) glycosidic bond and a p -(1 ,4) glycosidic bond.
  • the main component present in this extract is well represented by uronic acid (the main part is glucuronic acid) and hydroxyl acid [doi. org/10.1016/j.fshw.2021.04.009], which are important to stabilise the extract for a long time without significant changes on its physical and chemical properties, such as electrical conductivity or pH.
  • TFPS is extracted from the spores, mycelia and fermentation fluid of Tremella fuciformis by different extraction methods, including hot water extraction, alkali extraction, sonication-assisted extraction, cold water extraction and others [doi: 10.1016/bs.pmbts.2019.03.002]; these different experimental conditions can result in different polysaccharide fractions, leading to different CAS numbers.
  • Greeniuronic/Greendroitin the production process starts from the spores and involves several steps necessary to obtain an extract through extraction, purification and refining by means of alcohol solution, sieving and crushing [https://doi.org/10.3390/ijms23158114].
  • our extraction process uses the enzyme MetarhiziumtaiiGYYA0601 CGMCCNO.2880, which does not alter the p-1,4-D-glucuronic acid bond leading to hyaluronidase, preserving the presence of this monosaccharide.
  • this method removes excess monosaccharides (such as mannose, xylose, fucose, galactose, glucose%) and purifies the Tremella fuciformis sample allowing only GalNAc and GlcA (hence HA and CS) to be obtained.
  • HA or CS in TFPS.
  • HA was detected using the corresponding disaccharide ADi-HA generated by the enzymatic hydrolysis of chondroitinase AC of Tremella fuciformis extract.
  • ADi-HA generated by the enzymatic hydrolysis of chondroitinase AC of Tremella fuciformis extract.
  • the identity of this disaccharide was established by comparison with the reference standard ADi- HA and by the protonated and sodium positive ions detected in its mass spectrum. Furthermore, the same analysis revealed the absence of chondroitin monosulphates 4 and 6, which were eluted at 6.25 and 5.35 minutes, respectively.
  • Galla et al New hyaluronic acid from plant origin to improve joint protection— An in vitro study (ij ms-23-08114-v2) published on July 23, 2022, and Galla et al: In vitro analysis of the effects of plant-derived chondroitin sulfate from intestinal barrier to chondrocytes (1-s2.0- S176464622003553-main) published on October 17, 2022.
  • WC2020/245809 published on December 10, 2020, and WC2021/250566 published on December 16, 2021. All these 4 publications are herewith incorporated by reference.
  • FIG. 1 HPLC-UV and high-resolution mass spectrometry (HRMS) analysis of Greenluronic® after enzymatic hydrolysis with chondroitinase AC.
  • HRMS mass spectrometry
  • FIG. 1 An example of HA molecular weight determination on 1 % Agarose gel.
  • FIG. 3 Cell viability and ROS production on CaCo-2 cells.
  • panel (A) and (B) dose-response study on cell viability measured by MTT test of both Greenluronic® and Sodium Hyaluronate from 2h to 6h.
  • panel (C) and (D) ROS production of both Greenluronic® and Sodium Hyaluronate measured by reduction of cytochrome C from 2h to 6h.
  • Data are mean ⁇ SD of five independent experiments performed in triplicates vs control values (0% line).
  • FIG. 4 Permeability study on CaCo-2 cells.
  • A TEER Value using EVOM3; from (B) to (D) the analysis of TJ measured by Enzyme-Linked Immunosorbent Assay (ELISA) test (Occludin, Claudinl and Zo1 , respectively); in (E) the Papp values in which data ⁇ 0.2 x 10-6 cm/s means very poor absorption with a bioavailability ⁇ 1%, data between 0.2x10-6 and 2x10-6 cm/s with bioavailability between 1 and 90%, and data >2 x 10-6 cm/s means very good absorption with a bioavailability over 90%.
  • F HA quantification measured by ELISA kit.
  • FIG. 6 Greenluronic® and Sodium Hyaluronate effects on T/C-28a2 cells during OA conditions.
  • A mitochondrial metabolism tested by MTT test; in (B) ROS production; in (C) NFkB analysis by ELISA test; in (D) proliferation analysis by crystal violet and in (E) HA quantification by ELISA kit.
  • Data are mean ⁇ SD of five independent experiments performed in triplicates expressed as per-centage compared to control (0% line). * p ⁇ 0.05 vs control; y p ⁇ 0.05 vs 10
  • FIG. 7 Analysis of the main intracellular pathways activated in T/C-28a2 cells during AO conditions. The results demonstrated a reduction of apoptotic pathways and an improving of the survival pathways supporting the ability of HA to restore the OA damage.
  • A BAX activity
  • B Caspase 9 activity
  • C ERK/MAPK activity. All these results are obtained from specifically ELISA kit. Data are mean ⁇ SD of five independent experiments performed in triplicates compared to control value (0% line). * p ⁇ 0.05 vs control; y p ⁇ 0.05 vs 10
  • FIG. 8 Western blot and densitometric analysis of the main intracellular pathways activated in T/C-28a2 cells during AO conditions.
  • A OPG activity measured by ELISA test
  • B the CD44
  • C cyclin D1 densitometric analysis of the specific Western blot which are reported as an example in (D).
  • Data are mean ⁇ SD of five independent experiments performed in triplicates compared to control value (0% line). * p ⁇ 0.05 vs control; y p ⁇ 0.05 vs 10pig/ml of LPS; # p ⁇ 0.05 vs Sodium Hyaluronate 1 pig/pil.
  • Figure 10 the process for preparing a powder of HMWHA.
  • Figure 11 the analysis of Greenluronic® performed with 1 H MAS NMR.
  • the Applicant has carried out the characterization of Greenluronic®, in particular a High Performance Liquid Chromatography (HPLC) analysis has been made.
  • HPLC High Performance Liquid Chromatography
  • the high molecular weight of hyaluronic acid or salt thereof (HMWHA) of plant origin (called Geenluronic®) of the present invention may comprise or, alternatively, consist of disaccharide ADi-HA generated by chondroitinase AC enzymatic hydrolysis of the Tremella extract.
  • the identity of this disaccharide was established by comparison with the ADi-HA reference standard and by the protonated and sodiate positive ions detected in its mass spectrum.
  • the same analysis also revealed the absence of chondroitin 4 and 6 mono-sulfates which eluted at 6.25 and 5.35 min respectively.
  • the high molecular weight of hyaluronic acid or salt thereof (HMWHA) of plant origin (called Geenluronic®) of the present invention may comprise or, alternatively, consist of disaccharide ADi-HA generated by chondroitinase AC enzymatic hydrolysis of the Tremella extract, more preferably it does not contain (absence of) chondroitin 4 and 6 mono-sulfates.
  • T able 1 Quantification of HA.
  • the high molecular weight of hyaluronic acid or salt thereof (HMWHA) of plant origin (called Geenluronic®) of the present invention may have a content of glucuronic acid comprised in a range from 80% to 98%, preferably from 85% to 95%, for example 90%.
  • the high molecular weight of hyaluronic acid or salt thereof (HMWHA) of plant origin (called Geenluronic®) of the present invention may comprise or, alternatively, consist of disaccharide ADi-HA generated by chondroitinase AC enzymatic hydrolysis of the Tremella extract, preferably it does not contain (absence of) chondroitin 4 and 6 mono-sulfates, more preferably it may further have a content of glucuronic acid comprised in a range from 80% to 98%, preferably from 85% to 95%, for example 90%.
  • the high molecular weight of hyaluronic acid or salt thereof (HMWHA) of plant origin (called Geenluronic®) of the present invention may further have an average molecular weight comprised in a range from about 1500 kDa to about 20000 kDa, preferably from about 1650 kDa to about 15000kDa, more preferably from about 2000 kDa to about 10000 kDa, for example about 3000 kDa, about 4000 kDa, about 5000 kDa, about 6000 kDa, about 7000 kDa, about 8000 kDa, or about 9000 kDa.
  • the high molecular weight of hyaluronic acid or salt thereof (HMWHA) of plant origin (called Geenluronic®) of the present invention: a. may comprise a disaccharide ADi-HA generated by chondroitinase AC enzymatic hydrolysis of the Tremella extract, b. does not contain (absence of) chondroitin 4 and 6 mono-sulfates, c. may further have a content of glucuronic acid comprised in a range from 80% to 98%, preferably from 85% to 95%, for example 90%, d.
  • an average molecular weight comprised in a range from about 1500 kDa to about 20000 kDa, preferably from about 1650 kDa to about 15000kDa, more preferably from about 2000 kDa to about 10000 kDa, for example about 3000 kDa, about 4000 kDa, about 5000 kDa, about 6000 kDa, about 7000 kDa, about 8000 kDa, or about 9000 kDa.
  • the Applicant has also performed a dose-response and time-course study of Greenluronic® on CaCo-2 cells.
  • the human immortalized colorectal adenocarcinoma (CaCo-2) cell line was used to perform a dose-response study to exclude any cytotoxic effects.
  • the analysis was performed comparing the effects of Greenluronic® to sodium hyaluronate testing them at the same concentration (ranging from 0.125 to 1 pg/pL) on cell viability and ROS production in CaCo-2 cells in a timecourse study (from 2 h to 6 h).
  • the cell viability of the CaCo-2 cells measured by 3-(4, 5-D imethy lthiazol-2-y l)-2,5- diphenyltetrazolium bromide (MTT) assay, showed time and concentration dependent effects of both substances (Figure 3A), and the beneficial effects compared to control (p ⁇ 0.05) were maintained during all periods of stimulation excluding any cytotoxic effect at all dosage tested.
  • the Applicant has also carried out a permeability analysis of Greenluronic® using an in vitro model of intestinal barrier.
  • permeability to obtain additional information about the Greenluronic® intestinal absorption, further experiments were carried out performing a 3D in vitro model in order to mimic the in vivo complexity of the intestinal barrier.
  • 1 pig/pil Greenluronic® and 1 pig/pil sodium hyaluronate were tested from 2h to 6h in order to measure transepithelial electrical resistance (TEER) values, the apparent permeability coefficient (Papp) values and the HA concentration to predict their bioavailability.
  • TEER transepithelial electrical resistance
  • Papp apparent permeability coefficient
  • the data obtained show that the intestinal adsorption has a physiological trend as can be observed from the analysis of TEER and tight junction (TJ).
  • TJ tight junction
  • the passage through the intestinal epithelium demonstrates that both sodium hyaluronate and Greenluronic® were able to maintain the epithelial integrity increasing the ionic flux of the paracellular exchanges across the intestinal epithelial compared to control (p ⁇ 0.0001).
  • Greenluronic® demonstrates the better effect compared to sodium hyaluronate during all time of the stimulation (p ⁇ 0.0001 ) as reported in Figure 4A.
  • Greenluronic® was able to counteract these negative effects caused by LPS alone (p ⁇ 0.05) better than sodium hyaluronate (about 47% on cell viability and 2 time on ROS production respectively, p ⁇ 0.05).
  • NFkB nuclear factor kappa B
  • 1 pig/pil Greenluronic® was able to reduce the negative effect produced by LPS (about 1.25 times more) better than sodium hyaluronate (about 50%, p ⁇ 0.05).
  • This recovery mechanism was confirmed also by proliferation assay (panel 6D) in which T/C-28a2 cells lost their proliferative properties when treated only with 1 Opig/ml of LPS (p ⁇ 0.05 compared to control).
  • Greenluronic® exerted the main effects compared to sodium hyaluronate on Bax (about 2 time less, p ⁇ 0.05) and Caspase-9 activities (about 1.5 time less, p ⁇ 0.05) suggesting that Greenluronic® contributes to cell protection.
  • both 1 pig/pil Greenluronic® and sodium hyaluronate were able to reduce the damage induced by 10 pig/ml of LPS (p ⁇ 0.05), confirming the positive role of HA contained in two agents in stimulating chondrocytes metabolism.
  • 1 pig/ml Greenluronic® appears to be able to induce main effects compared to sodium hyaluronate (p ⁇ 0.05) to counteract the negative effects of OA inductor.
  • 1 pig/ml Greenluronic® is able to restore the damage induced by 10pig/ml of LPS in all parameters tested (about 60% for OPG, one time more for CD44 and 57% for cyclin D1 expression, p ⁇ 0.05), suggesting that it could ameliorate chondrocyte pathological conditions by activating them through the markers responsible for articular joint homeostasis.
  • HA is the useful tool in the management of patients with OA, since clinical data indicate its ability to reduce pain and improve joint function, with a potential ability to modify chondrocytes activity [29], It should also be taken into account that the administration of HA by intraarticular injection can also cause adverse effects such as infectious arthritis and cartilage damage [Zhang Y, Chen X, Tong Y, Luo J, Bi Q. Development and Prospect of Intra-Articular Injection in the Treatment of Osteoarthritis: A Review. J Pain Res. 2020 Aug 4;13:1941-1955. doi: 10.2147/JPR.S260878.].
  • Greenluronic® contains a large amount of HA with a chemical profile useful to be a new nutraceutical product.
  • Greenluronic® has a higher amount of HA that reaches the plasma level compared to control (p ⁇ 0.05) and compared to sodium hyaluronate within 4h and 5h, confirming the hypothesis that Greenluronic® improves the absorption during physiological time of intestinal digestion and improving its bioavailability.
  • Greenluronic® treatment indicated that a substantial part of HA is absorbed without damaging the intestinal epithelium; this is a crucial point since HA has a role in decreasing the permeability by enhancing the tight junction proteins.
  • dysbiosis-related gut permeability determined lower mRNA levels of TJ, ZO-1 and occludin, and higher LPS plasma levels in vivo model which has a positive association of synovial LPS with inflammation and disease severity in articular chondrocytes in OA patients [Huang ZYu, Chen J, Li BLei, et al.. Faecal microbiota transplantation from metabolically compromised human donors accelerates osteoarthritis in mice. Ann Rheum Dis. 2020;79(5):646- 656; Kolasinski SL, et al..
  • Greenluronic® was able to stimulate cell viability and induce chondrocyte proliferation without causing adverse effects, also compared to conventional HA supplementation. Indeed, the beneficial effects of Greenluronic® due to the presence of HMWHA into chondrocytes activity supporting the hypothesis of this use on joint-inflammatory conditions. Since OA is a disease of the whole joint and a multifactorial entity, there are various therapeutic strategies that involve numerous fields of medicine: rheumatology, orthopedics, geriatrics, psychiatry, general practitioners and physiotherapists. The goal of OA therapy is to reduce pain and increase patients' quality of life.
  • HA has shown not only beneficial effects on articular cartilage trophism, but also antinociceptive effects with a significant reduction in pain [Jimbo S, Terashima Y, Teramoto A, Takebayashi T, Ogon I, Watanabe K, Sato T, Ichise N, Tohse N, Yamashita T. Antinociceptive effects of hyaluronic acid on monoiodoacetate-induced ankle osteoarthritis in rats. J Pain Res. 2019 Jan 3; 12: 191-200.
  • Greenluronic® is obtained from White Tremella (Silver Ear), which is a traditional foodstuff with medicinal application in China [39], The production process involves several steps necessary to obtain a final extract and includes a new technology based on patent N°WO2021/250566 and N°WC2020/245809 from Vivatis Pharma GmbH, Gruner Deich 1-3, 20097 Hamburg, Germany.
  • the process involves steps of extraction, purification, refining by alcohol solution, sieving and crushing.
  • the resulting powder is then packed and tested for metals and stored [40, 41],
  • the sodium hyaluronate (Merck Life Science, Rome, Italy) was tested to verify the mechanism of action of Greenluronic®.
  • DMEM Dulbecco's Modified Eagle's Medium
  • FBS foetal bovine serum
  • 2 mM L-glutamine Merck Life Science, Rome, Italy
  • 1 % penicillinstreptomycin Merck Life Science, Rome, Italy
  • HA was also confirmed by HPLC (Shimadzu, Kyoto, Japan) analysis according to the method reported in literature [42], as described in Appendix A (Supplementary Material 1 and 2). Briefly; 20 pL of TRIS buffer (3.0 g TRIZMA base, 4.0 g sodium acetate trihydrate, 1.46 g sodium chloride, and 50 mg crystalline bovine serum albumin dissolved in 100 mL of 0.12 M HCI, pH 7.3 with 6 M HCI.
  • TRIS buffer 3.0 g TRIZMA base, 4.0 g sodium acetate trihydrate, 1.46 g sodium chloride, and 50 mg crystalline bovine serum albumin dissolved in 100 mL of 0.12 M HCI, pH 7.3 with 6 M HCI.
  • the assay performed to quantify the concentration of HA on material samples was the same reported in literature [43], Briefly, 1 mg of raw sample was dissolved in 1 mL of deionized water and 200pil of resuspended samples were displaced in new eppendorf, diluted with 1.2 mL of sulfuric acid (Merck Life Science, Rome, Italy) with 0.0125M tetraborate (Merck Life Science, Rome, Italy), shaked for 20 seconds and then boiled at 100°C for 5 min.
  • agarose gel The determination of the molecular weight of HA before exploring its biological effects, was carried out using 1% agarose gel, following the method reported in literature [45], Briefly, 0.3 g agarose (Merck Life Science, Rome, Italy) was dissolved in 30ml of Tris-acetate-EDTA (TAE) buffer (48.5g tris base, 11.4 mL acetic acid and 0.5 M EDTA pH 8, all substances were purchased from Merck Life Science, Rome, Italy) and the solution was heated for 30 seconds in a microwave at high power. The gel was poured into the holder and allowed to solidify before performing a pre-run at 100V for 45 min, using the Mini-Sub Cell GT System (Bio-Rad, Hercules, California, USA).
  • TAE Tris-acetate-EDTA
  • samples were prepared dissolving 200pig of raw samples in 16 l of TAE buffer 1x.
  • 4 l of loading buffer (0.2% Bromophenol Blue, 1 ml of TAE 1x and 8.5ml of glycerol which were purchased from Merck Life Science, Rome, Italy) were added to each sample and to the molecular weights (mixture of 5 l of Select-HA HILadder and 5 l Select-HA Mega Ladder, Echelon Biosciences, Tebu-Bio srl, Magenta, Italy).
  • the samples were run at 100V until the samples reached 1cm from the end of the gel.
  • the gel was hydrated in H2O for 24h at room temperature in agitation and then the gel was placed in 30% ethanol with 0.015% Stains All dye (Merck Life Science, Rome, Italy) for 24 h in the dark.
  • the gel was decoloured for 30 min in H2O in the dark before proceeding with image acquisition using ChemiDocTM Touch Imaging System (Bio-Rad, Hercules, California, USA).
  • the image obtained was analyzed by Image Lab 3.0 software (Bio-Rad Hercules, California, USA).
  • the human epithelial intestinal cells purchased from the American Type Culture Collection (ATCC), were cultured in Dulbecco's Modified Eagle's Medi-um/Nutrient F-12 Ham (DMEM-F12, Merck Life Science, Rome, Italy) containing 10% FBS (Merck Life Science, Rome, Italy), 2 mM L-glutamine and 1% penicillin- streptomycin (Merck Life Science, Rome, Italy) maintaining in an incubator at 37°C and 5% CO2 [33], The cells used for the experiments were at passage numbers between 26 to 32 in order to preserve the integrative of paracellular permeability and transport properties [46] maintaining the similarity to the intestinal absorption mechanism following oral intake in humans.
  • ATCC American Type Culture Collection
  • the cells were plated in different manner to perform several experiments including 1 x104 cells in 96 well plates to study cell viability by MTT-based In Vitro Toxicology Assay Kit (Merck Life Science, Rome, Italy) and ROS production using cytochrome C (Merck Life Science, Rome, Italy) in a complete medium. 8h before the stimulation the cells were incubated with DMEM without red phenol and supplemented with 0.5% FBS (Merck Life Science, Rome, Italy), 2 mM L-glutamine and 1 % penicillin-streptomycin (both from Merck Life Science, Rome, Italy) at 37 °C to synchronize them.
  • the immortalized human juvenile costal chondrocyte cell line T/C-28a2 (purchased from Merck Life Science, Rome, Italy) was cultured in DMEM-F12 medium supplemented with 10% FBS (Merck Life Science, Rome, Italy), 2mM L- glutamine (Merck Life Science, Rome, Italy), and antibiotics (50 Ul/ml penicillin and 50 pg/ml streptomycin, Merck Life Science, Rome, Italy)) and maintained in an incubator at 5% CO2 and 95% humidity [54],
  • This cell line is representative and the most commonly used cells for mimicking joints [55] and they were used between passages 3 and 10 [56].
  • 1 xi o 4 cells were seeded in 96 well plates to study cell viability by MTT-based In Vitro Toxicology Assay Kit (Merck Life Science, Rome, Italy), ROS pro-duction using cytochrome C (Merck Life Science, Rome, Italy) and Crystal Violet (Merck Life Science, Rome, Italy) in a complete medium; additionally
  • T/C-28a2 a chondrocyte cell line widely used to study articular joints, were treated for 3 days [58] and, at the end of stimulation, the mitochondrial metabolism, cell proliferation, ROS production and hyaluronic acid quantification were tested. Finally, in order to mimic OA conditions, further experiments were performed pre-treating T/C-28a2 with 1 Opig/ml of LPS (Merck Life Science, Rome, Italy) for 24h [36] and then stimulating with Greenluronic® and sodium hyaluronate for 3 days to evaluate if they are able to restore the damage. In these conditions, the survival mechanisms and articular recovery were investigated.
  • the analysis of cell viability was performed using a classical technique based on MTT-based In Vitro Toxicology Assay Kit (Merck Life Science, Rome, Italy) [59], following the manufacturer's instructions. Indeed, at the end of stimulation, the cells were incubated with 1 % MTT dye for 2 h in an incubator at 37°C, 5% CO2, and 95% humidity, and then the purple formazan crystals were dissolved in equal volume of MTT Solubilization Solution. The absorbance was analyzed by spectrophotometer (In-finite 200 Pro MPlex, Tecan, Mannedorf, Switzerland) at 570 nm with correction at 690 nm, and results were expressed compared to the control (0% line) which represented untreated cells. The results reported an increase in the percentage of viable cells compared to the control and indicate a higher number of viable cells plus the control. This strategy can lead to a high level of safety of the stimulation and consequently to a correct analysis of the results.
  • TEER values were determined with EVOM3, coupled with STX2 chopstick electrodes (World Precision Instruments, Sarasota, FL, USA); this assay was carried out every 2 days for 21 days until reaching a TEER value > to 400 Qcm2 before the stimulation [33, 60], time required for the cell monolayer formation, for cell differentiation and for the exposition of the intestinal villi.
  • Donor dQ amount of substance transported (nmol or pg); dt: incubation time (sec); mO: amount of substrate applied to donor compartment (nmol or pg);
  • A surface area of Transwell membrane (cm 2 ); VDonor: volume of the donor compartment (cm 3 ).
  • the Human Occludin ELISA kit (OCLN kit, MyBiosource, San Diego, CA, USA) analyzed the occludin presence in CaCo-2 cell lysates, according to the manufacturer's instruction [33], Briefly, CaCo-2 cells were lysed with cold Phosphate Buffered Saline (PBS, Merck Life Science, Rome, Italy) 1 x, centrifuged at 1500 g for 10 min at 4 °C and 100 pL of each sample was transferred to the strip well before the incubation at 37 °C for 90 min.
  • PBS cold Phosphate Buffered Saline
  • the supernatants were removed, and the strips were incubated with 100 pL of Detection Solution A for 45 min at 37 °C; then, the strips were washed with Wash Solution and incubated with 100 pL of Detection Solution B for additional 45 min. At the end of this time, 90 pL of Substrate Solution was added followed by an incubation for 20 min at 37 °C in the dark, and then 50 pL of Stop Solution was used to block the enzymatic reaction. The plate was analyzed by a spectrophotometer (Infinite 200 Pro MPlex, Tecan, Mannedorf, Switzerland) at 450 nm. The concentration is expressed as pg/mL compared to a standard curve (range from 0 to 1500 pg/ml) and the results are expressed as percentage (%) versus control (0 line).
  • the Human Claudin 1 was measured in CaCo-2 lysates by ELISA kit (Cusabio Technology LLC, Huston, USA), following the manufacturer's instructions [33], Briefly, the cells were lysed with cold PBS 1 x (Merck Life Science, Rome, Italy) and centrifuged at 1500* g for 10 min at 4 °C. 100 pL of each sample was added to ELISA plate and incubated at 37 °C for 2h; then, the plate was washed and 100 pL of Biotin-antibody were added to the wells and incubated for 1 h at 37 °C.
  • the Human Tight Junction Protein 1 ELISA kit (MyBiosource, San Diego, CA, USA) was measured in CaCo-2, following the manufacturer's instructions [33], Briefly, the cells were rinsed with ice-cold PBS 1x (Merck Life Science, Rome, Italy) and processed with two freeze-thaw cycles; then cell lysates were centrifuged for 5 minutes at 5000xg at 4°C. 10Opil of each sample were collected and incubated on the ELISA plate at 37 °C for 90 min; after washing, 100 pL of Detection Solution A were added to each well and incubated for 45 min at 37 °C. The wells were washed and 100 pL Detection Solution B was added to the samples.
  • the cells were fixed with 1 % glutaraldehyde (Merck Life Science, Rome, Italy) for 15 min at room temperature, washed, stained with 10Opil 0.1 % aqueous crystal violet (Merck Life Science, Rome, Italy) for 20 min at room temperature and solubilized with 10Opil 10% acetic acid before reading the absorbance at 595 nm using a spectrophotometer (Infinite 200 Pro MPlex, Tecan, Mannedorf, Switzerland). The estimated number was determined comparing data to the control cells normalized to TO (measurement at the beginning of the stimulation) [61], The results are expressed as percentage (%) versus control (0 line).
  • the quantification of superoxide anion release was obtained following a standard protocol based on the reduction of cytochrome C [61], and the absorbance in culture supernatants was measured at 550 nm using the spectrophotometer (Infinite 200 Pro MPlex, Tecan, Mannedorf, Switzerland). The 02 rate was expressed as the mean ⁇ SD (%) of nanomoles per reduced cytochrome C per microgram of protein compared to the control (0 line) [61].
  • both cell types were lysed with 100 pi of cold PBS1x to measure the total HA following the instructions of Hyaluronic Acid ELISA Kit (Clue-Clone). Briefly, 50 pil of sample and reagent A were added in each well and after gently shaking the plate was incubated for 1 h at 37°C. At the end, the wells were washed three times and 100 pil of reagent B were added before incubating the plate for 30 minutes at 37°C, then 90 pil of substrate solution were added before incubating the plate for 20 minutes at 37°C.
  • ERK/MAPK activity was performed using the InstantOneTM ELISA (Thermo Fisher, Milan, Italy) on chondrocytes lysates [64], Briefly, 50 pL of lysate samples prepared in Lysis Buffer were tested in ELISA microplate strips after the incubation for 1 h at room temperature in a microplate shaker pre-coated with the antibody cocktail. After that, the strips were incubated with the detection reagent for 20 min before stopping the reaction with stop solution. The absorbance was measured by a spectrophotometer at 450 nm (Infinite 200 Pro MPlex, Tecan, Mannedorf, Switzerland) and the results expressed as means ⁇ SD (%) versus control (0 line). 16. OPG activity
  • the OPG/TNFRSF11 B Duo Set (R&D Systems, Minneapolis, MN, USA) was applied according to the manufacturer's instructions to verify the OPG involvement [65], Briefly, 100 pL of samples or standards were added to the well and incubated for 2h at room temperature protected from light and, after washing, 100 pL of the Detection Antibody were added to each well and incubated as previously described. After 2h, 100 pL of the working dilution of Streptavidin-HRP A were added to each well and incu-bated for 20 minutes at room temperature.
  • the NF-kB (p65) Transcriptional factor Assay kit was carried out to analyze the NF-KB DNA binding activity, following the manufacturer's instruction (Cayman Chemical Company, Ann Arbor, Ml, USA) [66], The concentration was calculated by comparing results to the standard curve generated by NF-kB (p65) Transcriptional factor positive control (ranging from 0 to lOpil/well according to different scaled dilution) and re-ported as means ⁇ SD (%) compared to control (0 line).
  • BAX activity was determined in chondrocytes lysates using ELISA kit (Human Bax ELISA Kit, MyBiosource, San Diego, CA, USA) according to the manufacturer's instruction [67], The absorbance of the samples was measured at 450 nm by a spectrophotometer (Infinite 200 Pro MPlex, Tecan, Mannedorf, Switzerland) and the results were compared to the standard curve (range from 0 to 2000 pg/ml) and expressed as means ⁇ SD (%) normalized to control value (0 line).
  • the Caspase 9 activity was investigated in chondrocytes lysates by ELISA kit (Caspase 9 Human ELISA Kit.Thermoscientific, Waltham, Massachusetts, USA), according to the manufacturer's instructions, reading the sample's absorbance at 450 nm with a spectrometer (Infinite 200 Pro MPlex, Tecan, Mannedorf, Switzerland). The data were obtained by comparing to a standard curve (ranging from 1.6 to 100 ng/ml) and the results are expressed as means ⁇ SD (%) compared to control value (0 line) [68],
  • chondrocytes were washed with ice-cold PBS 1X (Merck Life Science, Rome, Italy), and lysed using Complete Tablet Buffer (Roche, Ba-sel, Switzerland) supplemented with 2mM sodium orthovanadate (Na3VO4), 1 mM phenylmethanesulfonylfluoride (PMSF) (Merck Life Science, Rome, Italy), 1 :50 mix Phosphatase Inhibitor Cocktail (Merck Life Science, Rome, Italy), and 1 :200 mix Protease Inhibitor Cocktail (Merck Life Science, Rome, Italy) to obtain a total protein extract which was centrifuged at 14.000 g for 20 min at 4 °C.
  • PMSF phenylmethanesulfonylfluoride
  • PMSF Phosphatase Inhibitor Cocktail
  • Protease Inhibitor Cocktail Merck Life Science, Rome, Italy
  • 35pig of proteins for each extract were resolved on 8% and 10% SDS-PAGE gel and transferred to a polyvinylidene difluoride (PVDF) membrane which was incubated overnight with the specific primary antibodies such as Cyclin D1 (1 :500, Santa Cruz, CA, United States) and CD44 (1 :500, Santa Cruz, CA, United States). All protein expressions were normalized and verified through p-actin detection (1 :5000, Merck Life Science, Rome, Italy), and ex-pressed as mean ⁇ SD (%) compared to control value (0 line).
  • PVDF polyvinylidene difluoride
  • Preferred embodiments of the present invention FRn are as follows.
  • HMWHA high molecular weight of hyaluronic acid or salt thereof (HMWHA) of plant origin present that comprises or, alternatively, consists of disaccharide ADi-HA obtained by chondroitinase AC enzymatic hydrolysis of the Tremella extract.
  • FR2 The mixture according to FR1 , wherein the high molecular weight of hyaluronic acid or salt thereof (HMWHA) of plant origin does not contain chondroitin 4 and 6 mono-sulfates.
  • FR3 The mixture according to FR1 o Fr2, wherein the high molecular weight of hyaluronic acid or salt thereof (HMWHA) of plant origin has a content of glucuronic acid comprised in a range from 80% to 98%, preferably from 85% to 95%, for example 90%.
  • HMWHA high molecular weight of hyaluronic acid or salt thereof
  • FR4 The mixture according to anyone of FR1-3, wherein the high molecular weight of hyaluronic acid or salt thereof (HMWHA) of plant origin has an average molecular weight comprised in a range from about 1500 kDa to about 20000 kDa, preferably from about 1650 kDa to about 15000kDa, more preferably from about 2000 kDa to about 10000 kDa, for example about 3000 kDa, about 4000 kDa, about 5000 kDa, about 6000 kDa, about 7000 kDa, about 8000 kDa, or about 9000 kDa.
  • HMWHA high molecular weight of hyaluronic acid or salt thereof
  • FR5 The mixture according to anyone of FR1-4, wherein said high molecular weight of hyaluronic acid or salt thereof (HMWHA) of plant origin has a higher amount of HA that crosses the barrier and reaches the plasma level compared to control (p ⁇ 0.0001) and compared to sodium hyaluronate (about 30%, p ⁇ 0.0001) with the greatest effects between 4h and 5h.
  • HMWHA high molecular weight of hyaluronic acid or salt thereof
  • FR6 The mixture according to anyone of FR1-5, wherein said high molecular weight of hyaluronic acid or salt thereof (HMWHA) of plant origin in an amount of 1 pig/pil is was able to reduce the negative effect produced by LPS (about 1.25 times more) better than sodium hyaluronate (about 50%, p ⁇ 0.05).
  • HMWHA high molecular weight of hyaluronic acid or salt thereof
  • a composition comprising a mixture according to anyone of FR1-7, and optionally at least one excipients or carrier of pharma or food grade, wherein said composition is for use:
  • composition according to FR8 wherein said composition is preferably a food or nutraceutical composition, more preferably for oral use.
  • HMWHA high molecular weight of hyaluronic acid or salt thereof
  • ADAMTS disintegrin and metalloproteinase with thrombospondin motifs
  • CaCo-2 the human immortalized colorectal adenocarcinoma cell line
  • CD44 differentiation cluster 44
  • DMEM/F12 Dulbecco's modified Eagle's medium/Nutrient F-12 Ham
  • ERK extracellular signal-regulated kinases
  • ERK/MAPK mitogen-activated protein kinases/extracellular signal-regulated kinase
  • FBS fetal bovine serum
  • FBS foetal bovine serum
  • GAGs glycosaminoglycan heteropolysaccharides family
  • HMWHA high-molecular weight HA
  • IBD inflammatory bowel disease
  • IL-10 interleukin (IL) -10
  • LMWHA low molecular weight HA
  • LPS lipopolysaccharide
  • MTT 3-(4,5-Dimethy I thiazol-2-y l)-2,5-diphenyl tetrazolium bromide
  • MMPs matrix metalloproteinases
  • MPK-1 mitogen-activated protein kinase phosphatase 1
  • Na3VO4 sodium orthovanadate
  • NFkB nuclear factor kappa B
  • NSAIDs non-steroidal anti-inflammatory drugs
  • NO nitric oxide (NO)
  • OPG osteoprotegerin
  • PBS phosphate buffered saline
  • PVDF polyvinylidene difluoride
  • RHAMM hyaluronan mediated motility receptors
  • ROS reactive oxygen species
  • TEER transepithelial electrical resistance
  • T/C-28a2 human chondrocyte cells
  • TJ tight junction
  • ZO-1 zonula occludens-1
  • Mobile phase B 340 mg of tetrabutylammonium bisulphate dissolved in 330 mL of water HPLC grade, then after the solution is at room temperature, brought to 1000 mL with acetonitrile.
  • nuclear magnetic resonance (NMR) spectroscopy is a powerful tool for this type of study, e.g. 13 C NMR measurements can reveal the structural and dynamical features of polysaccharides.
  • isotopic enrichment with 13 C is essential to apply advanced NMR experiments since the NMR-active isotopes, including 13 C, are naturally very low (1.1 % and 0.4%). https://doi.org/10.1016/bs.abr.2019.11.005.
  • NMR spectroscopy The area below the signals in 1 H NMR spectra is proportional to the number of protons responsible for the signal. It can obtain quantitative information about the material's chemical structure. DS of modified HA is usually calculated from the ratio of signal integrals of methyl protons of the N-acetyl residue of HA as backbone reference peak and specific signals of protons of the grafted moiety. Low-molecular-weight HA derivatives can be easily characterized by NMR spectroscopy due to the very low viscosity of the solutions. Still, the accuracy of DS determination by conventional 1D 1 H NMR is strongly limited in highly viscous environments, where interactions between polymer chains are very strong, and aggregation phenomena may occur.
  • NMR signals of specific groups are quantitative only when mobile, i.e., not implied in an ordered structure such as helical structures, in which hydrogen bonds can stabilize in stereoregular polymers or by specific interactions.
  • high molecular weight HA such as Greeniuronic®
  • spectra acquired at room temperature in D2O proton mobility of acetyl groups, backbone protons and anomeric protons varies between 41% and 56.4%. Increasing the temperature resulted in an increase of proton mobility to 74-83%, but it was observed that interactions between different chains were still present at 85 °C.
  • the graph in Figure 13 shows the same NMR spectrum as Greeniuronic: Greeniuronic® ppm 4.37 and HA ppm from bibliography 4.79. This slight difference in proton shift is due to the high molecular weight of Greeniuronic® (1650kDa) compared to HA from the bibliography, which has a molecular weight of 1200kDa.
  • High molecular weight hyaluronic acid down- regulates the gene expression of osteoarthritis-associated cytokines and enzymes in fibroblast-like synoviocytes from patients with early os-teoarthritis.
  • CircFADS2 is downregulated in osteoarthritis and suppresses LPS-induced apoptosis of chon-drocytes by regulating miR-195-5p methylation.

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Abstract

In recent decades, hyaluronic acid (HA) has attracted great attention as a new treatment option for osteoarthritis. Classical therapies are not able to stop the cartilage degeneration process nor do they favor tissue repair. Nowadays, it is accepted that high molecular weight HA can reduce inflammation by promoting tissue regeneration; therefore, the aim of this study was to verify the efficacy of a new high molecular weight HA of plant origin (called GreenIuronic®) in maintaining joint homeostasis and preventing the harmful processes of osteoarthritis. Methods: The bioavailability of GreenIuronic® was investigated in a 3D intestinal barrier model that mimics human oral intake while excluding damage to the intestinal barrier. Furthermore, the chemical significance and bio-logical properties of GreenIuronic® were investigated, even in conditions that simulate osteoarthritis. Results: Our data demonstrated that GreenIuronic® crosses the intestinal barrier without side effects as it has a chemical-biological profile, which could be responsible for many specific chondrocyte functions. Furthermore, in the osteoarthritis model, GreenIuronic® can modulate the molecular mechanism responsible for preventing and restoring the degradation of cartilage. Conclusion: According to our results, this new form of HA appears to be well absorbed and distributed to chondrocytes, preserving their biological activities. Therefore, the oral administration of GreenIuronic® in humans can be considered a valid strategy to obtain beneficial therapeutic effects during osteoarthritis.

Description

A new high molecular weight of hyaluronic acid or salt thereof of plant origin for use in maintaining joint homeostasis and preventing the harmful processes of osteoarthritis
The present invention relates to new high molecular weight of hyaluronic acid or salt thereof (HMWHA) of plant origin (called Geenluronic®) for use in maintaining joint homeostasis and preventing the harmful processes of osteoarthritis. Further, the present invention relates to a process for production of said new high molecular weight of hyaluronic acid or salt thereof (HMWHA) of plant origin (called Geenluronic®). Moreover, the present invention refers to a composition comprising a mixture that comprises or, alternatively, consists of said new high molecular weight of hyaluronic acid or salt thereof (HM HA) of plant origin (called Geenluronic®), preferably said composition and mixture is for use (I) in maintaining joint homeostasis and preventing the harmful processes of osteoarthritis, and/or (ii) in a method for preventing and restoring the degradation of cartilage, and/or (ill) in a method for the treatment of osteoarthritis.
STATE OF THE ART
Osteoarthritis (OA) is a slow progressive joint disorder which causes several disabilities in the adult population [1], For a long time, OA was regarded as a progressive wear of the joint cartilage alone. However, recent research has shown that it is an inflammatory disease of the entire synovial joint, which includes not only the mechanical degeneration of the articular cartilage but also the concomitant structural and functional change of the entire joint, including the synovium, meniscus, periarticular ligaments and subchondral bone [Loeser RF, Goldring SR, Scanzello OR, Goldring MB. Osteoarthritis: a disease of the joint as an organ. Arthritis Rheum. 2012 Jun;64(6): 1697-707. doi: 10.1002/art.34453. Epub 2012 Mar 5], An important role in infrapatellar fat pad inflammation and fibrosis has also recently been discovered [Belluzzi E, Macchi V, Fontanella CG, Carniel EL, Olivotto E, Filardo G, Sarasin G, Porzionato A, Granzotto M, Pozzuoli A, Berizzi A, Scion! M, De Caro R, Ruggieri P, Vettor R, Ramonda R, Rossato M, Favero M. Infrapatellar Fat Pad Gene Expression and Protein Production in Patients with and without Osteoarthritis. Int J Mol Sci. 2020 Aug 21 ;21 (17):6016. doi: 10.3390/ijms21176016], Today the treatment modalities for OA include non-pharmacological (e.g. physiotherapy), pharmacological (e.g. steroidal and non-steroidal-anti-inflammatory drugs) or intra-articular (e.g. injection of hyaluronic acid) therapies [3,4], These classical therapies can reverse the symptoms only in a small number of cases, but they don't stop the degeneration process of the cartilage or promote the repair of the tissue. Therefore, the development of new therapies is a primary goal, preferably hypothesizing the oral administration which remains the preferred route for a drug delivery due to its low invasiveness, high efficiency, and better patient compliance [5], Obviously in this case, the bio accessibility and bioavailability of orally administered compounds need to be investigated in a preclinical model in order to evaluate the ability to cross the intestinal barrier after oral administration [6], During the last decades, hyaluronic acid (HA) has attracted great attention as a new treatment option for knee OA pain [2, 7, 8], HA is a natural polymer belonging to the glycosaminoglycan heteropolysaccharides family (GAGs), but unlike these molecules it is not sulphated, and it is not synthesized by Golgi enzymes [8], In addition, the native form appears as a very long polymer, called high-molecular weight HA (HMWHA) [8], Therefore, the native HA consists of 2000- 25000 disaccharide units, corresponding to 106-107 Da molecular weight; for that a long chain contains more than 10000 units like -4000 kDa [7, 9], In the biological systems, HMWHA (also called as native HA) is degraded into small fragments named low molecular weight HA (LMWHA) corresponding to different molecular weight; in particular HMWHA has >1-10 MDa; intermediate HA has >100-1000 kDa and LMWHA has the molecular weight between 1 and 10 kDa [7, 10], Several studies reported that the structural and biological properties of HA within medical, pharmaceutical, and cosmetic applications analyzing the role of HA in inflammation and tissue regeneration are related to its specific molecular weight [11 , 12], Applications of HA depend on its biological effects on cell differentiation and proliferation, and on its ability to lubricate, hydrate, and interact with various receptors present on the cell surface. It is this interaction that facilitates the exact delivery of drugs, facilitating their internalization in target sites. The safety, tolerability, and efficacy of HA-based formulations for the treatment of various types of joint diseases have been validated in several studies [8, 13], It is widely accepted that exogenous hyaluronic acid is incorporated into articular cartilage where it may have a direct biological effect on chondrocytes to improve joint lubrication as well de-scribed by clinical studies. The concept of viscosupplementation is based upon the hypothesis that HA administration could improve the rheological properties of joints, promote the endogenous synthesis of a HMWHA and possibly more functional HA, thereby improving mobility, and articular function, and decreasing pain. The growing use of HA in medical practise can be explained by their effectiveness and versatility as well as their favorable safety profiles [Urdiales-Galvez F, Delgado NE, Figueiredo V, et al. Treatment of Soft Tissue Filler Complications: Expert Consensus Recommendations. Aesthetic Plast Surg. 2018; 42(2): 498-510. doi: 10.1007/s00266-017-1063-0], Nowadays, sodium HA seems to be the best choice, available on market, since it exerts analgesic effect by blocking pain receptors in synovial tissues and holding endogenous pain sub-stances in its molecule [Gupta RC, Lail R, Srivastava A, Sinha A. Hyaluronic Acid: Molecular Mechanisms and Therapeutic Trajectory. Front Vet Sci. 2019;6:192. Published 2019 Jun 25. doi: 10.3389/fvets.2019.00192], However, it can be suggested that the characteristic steric configurations HMWHA are needed for the manifestation of the analgesic effect indicating possible clinical applications of all fragments of HA [Gupta RC, Lail R, Srivastava A, Sinha A. Hyaluronic Acid: Molecular Mechanisms and Therapeutic Trajectory. Front Vet Sci. 2019;6: 192. Published 2019 Jun 25. doi: 10.3389/fvets.2019.00192], Indeed, HA is one Food and Drug Administration (FDA) approved treatment for inflammatory conditions, including those affecting the joints, and is also acknowledged in Europe for its beneficial properties due to the therapeutic potential caused by native HA, but without toxicity [7], In particular, in vitro studies demonstrate that only native HA exerts an inhibitory effect on interleukin (IL) -1 p stimulated prostaglandin E2 (PGE2) production in inflammation-damaged bovine cartilage. This supports the hypothesis that HMWHA may be an important new strategy to restore proteoglycan content leading to a new cartilage protective strategy [7, 14], Synovial inflammation and structural and molecular changes of the joint system should be the target of OA therapies. However, currently, therapy with HA, a constituent of the synovial fluid and that has viscoelastic properties has been proposed [Galluccio F, Barskova T, Cerinic MM. Short-term effect of the combination of hyaluronic acid, chondroitin sulfate, and keratin matrix on early symptomatic knee osteoarthritis. Eur J Rheumatol. 2015;2(3): 106-108. doi: 10.5152/eurjrheum.2015.0019], Much research has been conducted on HA combined with anti-inflammatory drugs, via clinical trials or in vivo and in vitro studies, and the published data indicate with a good level of evidence that intra-articular injection of HA combined with antiinflammatory drugs can potentially relieve pain in OA knee patients [Euppayo, T., Punyapornwithaya, V., Chomdej, S. et al. Effects of hyaluronic acid combined with anti-inflammatory drugs compared with hyaluronic acid alone, in clinical trials and experiments in osteoarthritis: a systematic review and meta-analysis. BMC Musculoskelet Disord 18, 387 (2017). https://doi.org/10.1186/s12891-017-1743-6]. In the last years, symptomatic slow-acting drugs for OA have been vastly studied and many studies focused the attention on HA, or chondroitin sulfate (OS) combined with nonsteroidal anti-inflammatory drugs (NSAIDs) to limit the related adverse events in the gastrointestinal tract, kidney, and cardiovascular system [Galluccio F, Barskova T, Cerinic MM. Short-term effect of the combination of hyaluronic acid, chondroitin sulfate, and keratin matrix on early symptomatic knee osteoarthritis. Eur J Rheumatol. 2015;2(3): 106-108. doi:10.5152/eurjrheum.2O15.0019], Finally, to reduce the strong adverse effects due to drugs, HA may be combined with several agents including lactose-modified chitosan and cyclodextrin to improve chondroprotection and to stimulate cartilage growth reducing inflammation [Tarricone E, Elia R, Mattiuzzo E, et al. The Viability and An-ti-lnflammatory Effects of Hyaluronic Acid-Chitlac-Tracimolone Acetonide- p-Cyclodextrin Complex on Human Chondrocytes. Cartilage. 2021; 13(2_suppl):920S-924S. doi:10.1177/1947603520908658], Additional studies reported similar beneficial effects of HMWHA in OA and in other inflammatory conditions [15, 16], The main purpose induced by HMWHA is to promote chondroprotection and involves several proteins including binding to the receptor of the differentiation cluster 44 (CD44), which is required to inhibit the expression of IL-1 p, leading to a de-cline production of matrix metalloproteinases (MMPs) -1 , 2, 3, 9 and 13 [2, 17, 18], In addition, HA binds hyaluronan mediated motility receptors (RHAMM) to induce chondroprotection as well as CD44 binding [2], Regarding the mechanism activated by the binding of CD44, the most important is the role of the mitogen-activated protein kinase phosphatase (MKP) -1 which is able to inhibit the production of IL-1 p and consequently inhibit the MMPs within articular cartilage and finally prevent apoptotic events in the chondrocyte through the reduction of a disintegrin and metalloproteinase expression with thrombospondin motifs (ADAMTS) [2, 19, 20], Another important element is the production of reactive oxygen species (ROS) and nitric oxide (NO) which are normally involved in the apoptosis-dependent death of chondrocytes, leading to the degeneration of cartilage. In this context, the current literature reported that HMWHA after the binding with CD44 is able to prevent chondrocytes apoptosis by inhibiting PGE2 synthesis and interleukin activity such as IL-1p which is responsible of oxidative stress [2, 17, 21 , 22], Regarding the effectiveness of HA on joint tissue, it is important to remember that it can act as a passive structural molecule or exerts biological effects via a signaling molecule. Furthermore, since its different mechanism of action depends on the molecular weight, the link between the molecular weight and its pro and anti-inflammatory activities, the promotion and inhibition of the activation of migration and the blocking or promotion of the division is also important [8], Today some details are known about how HA exerts its different biological functions at various concentrations and molecular weights. For example, at the level of the intestinal mucosa, the intermediate HA and HMWHA have antioxidant and antimicrobial properties [23], Its importance is constantly growing because this substance has the ability to regulate tissue homeostasis and its physiological decrease is related to the aging process that leads to various diseases [23], Oral administration of exogenous HA has attracted the attention of researchers as a supplementary therapy to prevent or treat the aging process of cartilage and related diseases [Ewald CY (2021) Drug Screening Implicates Chondroitin Sulfate as a Potential Longevity Pill. Front. Aging 2:741843. doi: 10.3389/f rag i.2021.741843],
In view of the above, it is felt the need to have at our disposal a new HMWHA plant-derived HA which is able to maintain joint homeostasis in order to prevent all the harmful processes that can trigger the pathology of osteoarthritis OA.
In particular, it is felt the need to have at our disposal a composition comprising a mixture that comprises or, alternatively, consists of said new high molecular weight of hyaluronic acid or salt thereof (HMWHA) of plant origin for use (i) in maintaining joint homeostasis and preventing the harmful processes of osteoarthritis, and/or (ii) in a method for preventing and restoring the degradation of cartilage, and/or (iii) in a method for the treatment of osteoarthritis OA.
THE PRESENT INVENTION
The present invention relates to new high molecular weight of hyaluronic acid or salt thereof (HMWHA) of plant origin (called Geenluronic®) for use in maintaining joint homeostasis and preventing the harmful processes of osteoarthritis. Further, the present invention relates to a process for production said new high molecular weight of hyaluronic acid or salt thereof (HMWHA) of plant origin (called Geenluronic®). Moreover, the present invention refers to a composition comprising a mixture that comprises or, alternatively, consists of said new high molecular weight of hyaluronic acid or salt thereof (HMWHA) of plant origin (called Geenluronic®), preferably said composition and mixture is for use (i) in maintaining joint homeostasis and preventing the harmful processes of osteoarthritis, and/or (ii) in a method for preventing and restoring the degradation of cartilage, and/or (iii) in a method for the treatment of osteoarthritis.
The present invention refers to a mixture that comprises or, alternatively, consists of:
- a high molecular weight of hyaluronic acid or salt thereof (HMWHA) of plant origin present that comprises or, alternatively, consist of disaccharide ADi-HA obtained by chondroitinase AC enzymatic hydrolysis of the Tremella extract.
Preferably, the high molecular weight of hyaluronic acid or salt thereof (HMWHA) of plant origin does not contain chondroitin 4 and 6 mono-sulfates.
Preferably, the high molecular weight of hyaluronic acid or salt thereof (HMWHA) of plant origin has a content of glucuronic acid comprised in a range from 80% to 98%, preferably from 85% to 95%, for example 90%.
Preferably, the high molecular weight of hyaluronic acid or salt thereof (HMWHA) of plant origin has an average molecular weight comprised in a range from about 1500 kDa to about 20000 kDa, preferably from about 1650 kDa to about 15000kDa, more preferably from about 2000 kDa to about 10000 kDa, for example about 3000 kDa, about 4000 kDa, about 5000 kDa, about 6000 kDa, about 7000 kDa, about 8000 kDa, or about 9000 kDa. Preferably, said high molecular weight of hyaluronic acid or salt thereof (HMWHA) of plant origin has a higher amount of HA that crosses the barrier and reaches the plasma level compared to control (p<0.0001 ) and compared to sodium hyaluronate (about 30%, p<0.0001) with the greatest effects between 4h and 5h.
Preferably, said high molecular weight of hyaluronic acid or salt thereof (HMWHA) of plant origin in an amount of 1 pig/pil is was able to reduce the negative effect produced by LPS (about 1.25 times more) better than sodium hyaluronate (about 50%, p<0.05).
Preferably, said mixture is for use:
- (i) in a method for maintaining joint homeostasis and preventing the harmful processes of osteoarthritis, and/or
- (ii) in a method for preventing and restoring the degradation of cartilage, and/or
- (iii) in a method for the treatment of osteoarthritis OA.
The present invention also refers to a composition comprising said mixture and, optionally, at least one excipients or carrier of pharma or food grade, wherein said composition is for use:
- (i) in a method for maintaining joint homeostasis and preventing the harmful processes of osteoarthritis, and/or
- (ii) in a method for preventing and restoring the degradation of cartilage, and/or
- (iii) in a method for the treatment of osteoarthritis OA.
Preferably, said composition is preferably a food or nutraceutical composition, more preferably for oral use.
The present invention also refers to a process for preparing said mixture, wherein said process comprises steps of extraction of the high molecular weight of hyaluronic acid or salt thereof (HMWHA) from White Tremella (Silver Ear), purification, refining by alcohol solution, sieving and crushing to provide a powder of HMWHA.
For example, one embodiment of the process for preparing a powder of HMWHA is illustrated in Figure 10.
Glycosaminoglycans (GAGs) are linear polysaccharides formed from covalently bound disaccharide units. Their disaccharide repeating unit consists of an amino sugar (exoamines, including d-glucosamine and d-galactosamine) and a uronic acid (D-glucuronic acid and l-hyduronic acid). GAGs are generally covalently bound to a basic protein to form a proteoglycan with different physiological functions. They differ in chain length, protein binding, degree of sulphation and proportion of uronic acids, among others. They can be obtained with different structure and characteristics, such as sugar composition and degree of sulphation, depending on the extraction method and species of origin: for example, HA is a polysaccharide formed from long, repeated disaccharide units comprising N-acetyl-d-glucosamine (GalNAc) and d-glucuronic acid (GlcA) and is the only GAG that is not sulphated and not bound to proteins; in contrast, CS is a GAG formed from repeated disaccharides of GalNAc and GlcA with a shorter chain than HA (20-100 repeated units) with a structure that differs according to the different positions of the sulphate groups [https: //doi. org/10.1016/j.carbpol.2020 .116441], Consequently, several techniques have been developed and optimised to extract and differentiate HA and CS using detergents, enzymes and/or solvents to break down the structure and isolate GAGs from other complexes. In general, the methods are based on chemical hydrolysis of the tissue to ensure the breakdown of the proteoglycan core, followed by protein removal to recover the GAGs. The methods are based on the general steps of hydrolysis of the tissue, removal of impurities (such as proteins) and purification of HA and CS. Thus, GAGs differ in the amount of HA and CS recovered through the use of specific enzymes and/or solvents and also in the source of biomass used [https://doi.Org/10.1016/j.carbpol.2020.116441].
In the case of Greeniuronic/Greendroitin, which is the subject matter of the present invention, specific experimental conditions allow different fractions of the Tremella fuciformis polysaccharide (TFPS) to be obtained, which turns out to be a mixture of different polysaccharides with molecular weights ranging from 5.82 x 105 Da to 3.74 x 106 Da. The monosaccharides detected in TFPS include mannose, xylose, fucose, glucose, galactose and, in particular, glucuronic acid. In addition, TFPS is a mannitan formed by an a -(1 ,3) glycosidic bond and a p -(1 ,4) glycosidic bond. The main component present in this extract is well represented by uronic acid (the main part is glucuronic acid) and hydroxyl acid [doi. org/10.1016/j.fshw.2021.04.009], which are important to stabilise the extract for a long time without significant changes on its physical and chemical properties, such as electrical conductivity or pH.
Generally, TFPS is extracted from the spores, mycelia and fermentation fluid of Tremella fuciformis by different extraction methods, including hot water extraction, alkali extraction, sonication-assisted extraction, cold water extraction and others [doi: 10.1016/bs.pmbts.2019.03.002]; these different experimental conditions can result in different polysaccharide fractions, leading to different CAS numbers. In this specific case (Greeniuronic/Greendroitin), the production process starts from the spores and involves several steps necessary to obtain an extract through extraction, purification and refining by means of alcohol solution, sieving and crushing [https://doi.org/10.3390/ijms23158114]. In particular, our extraction process uses the enzyme MetarhiziumtaiiGYYA0601 CGMCCNO.2880, which does not alter the p-1,4-D-glucuronic acid bond leading to hyaluronidase, preserving the presence of this monosaccharide. In addition, this method removes excess monosaccharides (such as mannose, xylose, fucose, galactose, glucose...) and purifies the Tremella fuciformis sample allowing only GalNAc and GlcA (hence HA and CS) to be obtained. In addition, to differentiate the presence of HA or CS in TFPS, we performed the analysis with the HPLC method. The presence of HA was detected using the corresponding disaccharide ADi-HA generated by the enzymatic hydrolysis of chondroitinase AC of Tremella fuciformis extract. The identity of this disaccharide was established by comparison with the reference standard ADi- HA and by the protonated and sodium positive ions detected in its mass spectrum. Furthermore, the same analysis revealed the absence of chondroitin monosulphates 4 and 6, which were eluted at 6.25 and 5.35 minutes, respectively. However, due to the absence of the sulphate group in the disaccharide chondroitin 0 sulphate (ADOS), which results from the hydrolysis of chondroitin, the HPLC method, based on ion-pair retention, did not allow us to separate the disaccharides ADi-HA and AD-OS: in fact, they elute at 2.25 min [https://doi.org/10.3390/ijms23158114]. In addition, we compared our samples with a mixture of standard chondroitin disaccharides ADi-OS, ADI-4S, ADi-6S to confirm the absence of sulphate groups in the sample and therefore assumed the presence of only HA. At the same time, some experiments were conducted to verify the molecular weight and C4S/C6S content of Tremella fuciformis extract, comparing it with different forms of CS including plant-derived CS, market CS, chicken CS, shark CS and bovine CS as reported in the literature [https://doi.org/10.1016/jjff.2022.105285] indicating that these forms of CS are chemical analogues.
We make specific reference to the scientific articles Galla et al: New hyaluronic acid from plant origin to improve joint protection— An in vitro study (ij ms-23-08114-v2) published on July 23, 2022, and Galla et al: In vitro analysis of the effects of plant-derived chondroitin sulfate from intestinal barrier to chondrocytes (1-s2.0- S176464622003553-main) published on October 17, 2022. Moreover, we also make specific reference to the international patent applications WC2020/245809 published on December 10, 2020, and WC2021/250566 published on December 16, 2021. All these 4 publications are herewith incorporated by reference.
LIST OF FIGURES
Figure 1. HPLC-UV and high-resolution mass spectrometry (HRMS) analysis of Greenluronic® after enzymatic hydrolysis with chondroitinase AC. In (A) and in (B) HPLC-UV chromatogram of Greenluronic® sample and its positive HRMS spectrum. In (C) and in (D) HPLC-UV chromatograms of a mixture of chondroitin disaccharides standard ADi-OS, ADI-4S, ADI-6S and a solution of the disaccharide standard ADi-HA of HA.
Figure 2. In the figure an example of HA molecular weight determination on 1 % Agarose gel. The sample loads are described as follows by the abbrevations: MW = standard molecular weight Mega+HILadder specific for HA detection; L1= lane empty loaded with 10pil TAE buffer; L2= lane loaded with 100ug/10pil Sodium Hyaluronate; L3= lane empty loaded with 10pil TAE buffer; L4= lane loaded with 100ug/10pil Greenluronic®; L5= lane empty loaded with 10pil TAE buffer.
Figure 3. Cell viability and ROS production on CaCo-2 cells. In panel (A) and (B) dose-response study on cell viability measured by MTT test of both Greenluronic® and Sodium Hyaluronate from 2h to 6h. In panel (C) and (D) ROS production of both Greenluronic® and Sodium Hyaluronate measured by reduction of cytochrome C from 2h to 6h. Data are mean ± SD of five independent experiments performed in triplicates vs control values (0% line).
Figure 4. Permeability study on CaCo-2 cells. In (A) TEER Value using EVOM3; from (B) to (D) the analysis of TJ measured by Enzyme-Linked Immunosorbent Assay (ELISA) test (Occludin, Claudinl and Zo1 , respectively); in (E) the Papp values in which data <0.2 x 10-6 cm/s means very poor absorption with a bioavailability <1%, data between 0.2x10-6 and 2x10-6 cm/s with bioavailability between 1 and 90%, and data >2 x 10-6 cm/s means very good absorption with a bioavailability over 90%. In (F) HA quantification measured by ELISA kit. Data are mean ± SD of five independent experiments performed in triplicates. From B to D the means± SD are expressed comparing data to control value (0% line) and * p<0.05 vs control; # p<0.05 vs Sodium Hyaluronate 1 g/ l. On the contrary, in A, E and F the control sample are specifically reported and both Greenluronic and sodium hyaluronate are p<0.0001 vs control; # p<0.05 vs Sodium Hyaluronate 1 pig/pil. Figure 5. Analysis of Greenluronic® and Sodium Hyaluronate on human chondrocyte (T/C-28a2) cells functions. In (A) the mitochondrial metabolism tested by MTT test; in (B) the ROS production; in (C) the proliferation analysis by crystal violet stained and in (D) the HA quantification by ELISA kit. Data are expressed as mean ± SD compared to control (0% line) of five independent experiments performed in triplicates. * p<0.05 vs control; # p<0.05 vs Sodium Hyaluronate 1 pig/pil.
Figure 6. Greenluronic® and Sodium Hyaluronate effects on T/C-28a2 cells during OA conditions. In (A) mitochondrial metabolism tested by MTT test; in (B) ROS production; in (C) NFkB analysis by ELISA test; in (D) proliferation analysis by crystal violet and in (E) HA quantification by ELISA kit. Data are mean ± SD of five independent experiments performed in triplicates expressed as per-centage compared to control (0% line). * p<0.05 vs control; y p<0.05 vs 10|jg/ml of LPS; # p<0.05 vs Sodium Hyaluronate 1 pig/pil.
Figure 7. Analysis of the main intracellular pathways activated in T/C-28a2 cells during AO conditions. The results demonstrated a reduction of apoptotic pathways and an improving of the survival pathways supporting the ability of HA to restore the OA damage. In (A) BAX activity; in (B) Caspase 9 activity; in (C) ERK/MAPK activity. All these results are obtained from specifically ELISA kit. Data are mean ± SD of five independent experiments performed in triplicates compared to control value (0% line). * p<0.05 vs control; y p<0.05 vs 10|jg/ml of LPS; # p<0.05 vs Sodium Hyaluronate 1 pig/pil.
Figure 8. Western blot and densitometric analysis of the main intracellular pathways activated in T/C-28a2 cells during AO conditions. In (A) OPG activity measured by ELISA test, in (B) the CD44 and in (C) cyclin D1 densitometric analysis of the specific Western blot which are reported as an example in (D). Data are mean ± SD of five independent experiments performed in triplicates compared to control value (0% line). * p<0.05 vs control; y p<0.05 vs 10pig/ml of LPS; # p<0.05 vs Sodium Hyaluronate 1 pig/pil.
Figure 9. HA Production Flow Chart.
Figure 10: the process for preparing a powder of HMWHA.
Figure 11 : the analysis of Greenluronic® performed with 1 H MAS NMR.
Figure 12: 13C CPMAS NMR https://d .arq/10.1101/2023.03.16.532902
Figure 13: 1H NMR spectrum of high molecular weight HA
DETAILED DESCRIPTION OF THE PRESENT INVENTION
The Applicant has carried out the characterization of Greenluronic®, in particular a High Performance Liquid Chromatography (HPLC) analysis has been made.
The Applicant has found that the HPLC analysis of Greenluronic® (Figure 1) revealed the presence of HA which was detected as the corresponding disaccharide ADi-HA generated by chondroitinase AC enzymatic hydrolysis of the Tremella extract.
Preferably, the high molecular weight of hyaluronic acid or salt thereof (HMWHA) of plant origin (called Geenluronic®) of the present invention may comprise or, alternatively, consist of disaccharide ADi-HA generated by chondroitinase AC enzymatic hydrolysis of the Tremella extract. The identity of this disaccharide was established by comparison with the ADi-HA reference standard and by the protonated and sodiate positive ions detected in its mass spectrum. Moreover, the same analysis also revealed the absence of chondroitin 4 and 6 mono-sulfates which eluted at 6.25 and 5.35 min respectively.
Preferably, the high molecular weight of hyaluronic acid or salt thereof (HMWHA) of plant origin (called Geenluronic®) of the present invention may comprise or, alternatively, consist of disaccharide ADi-HA generated by chondroitinase AC enzymatic hydrolysis of the Tremella extract, more preferably it does not contain (absence of) chondroitin 4 and 6 mono-sulfates.
However, because of the absence of sulfate group in the di-saccharide chondroitin 0 sulfate (AD-OS), arising from chondroitine hydrolysis, the HPLC method, based on ion-pair retention, did not allow the separation between the disaccharides ADi-HA and AD-OS: indeed, they eluted at 2.25 min.
Moreover, since HPLC-UV analysis revealed a possible high concentration of HA in Greenluronic® sample, additional experiments were carried out to quantify the content of glucuronic acid in Greenluronic® and in sodium hyaluronate samples. As reported in Table 1 , the content of glucuronic acid of Greenluronic® is about 90% which is higher than that of sodium hyaluronate (about 62%). These data support what was observed in previous experiments in HPLC (reported above) about the purity of the Greenluronic® material.
Raw material Mean (%w/w) ± SD
Sodium Hyaluronate 62.5±2.121
Greenluronic® 90.5±6.364
T able 1 . Quantification of HA. The % w/w of all HA forms normalized on standard curves generated using glucuronic acid standard (ranging from 0 to 2mg/ml) analyzed at 340nm by spectrophotometry (Infinite 200 Pro MPlex, Tecan). Data are expressed as means ± standard deviation (SD) (%) of five independent experiments performed in triplicates.
Preferably, the high molecular weight of hyaluronic acid or salt thereof (HMWHA) of plant origin (called Geenluronic®) of the present invention may have a content of glucuronic acid comprised in a range from 80% to 98%, preferably from 85% to 95%, for example 90%.
Preferably, the high molecular weight of hyaluronic acid or salt thereof (HMWHA) of plant origin (called Geenluronic®) of the present invention may comprise or, alternatively, consist of disaccharide ADi-HA generated by chondroitinase AC enzymatic hydrolysis of the Tremella extract, preferably it does not contain (absence of) chondroitin 4 and 6 mono-sulfates, more preferably it may further have a content of glucuronic acid comprised in a range from 80% to 98%, preferably from 85% to 95%, for example 90%.
Finally, for the analysis of Greenluronic® size distribution, the agarose gel electrophoresis was used to define a range of the molecular weight. Agarose gel retards the electrophoretic mobility of HA molecules in a molecular weight dependent manner indicating that Greenluronic® may be considered the HMWHA (>1650kDa) as can be seen in Figure 2. On the contrary, the sodium hyaluronate has confirmed to be the lower-molecular weight HA (LMWHA) (300 kDa and 500 kDa). These results indicate that Greenluronic® molecular weight is higher than that of sodium hyaluronate and further experiments were performed in order to confirm the hypothesis that HMWHA exerts more beneficial effects compared to those of LMWHA [24],
Preferably, the high molecular weight of hyaluronic acid or salt thereof (HMWHA) of plant origin (called Geenluronic®) of the present invention may further have an average molecular weight comprised in a range from about 1500 kDa to about 20000 kDa, preferably from about 1650 kDa to about 15000kDa, more preferably from about 2000 kDa to about 10000 kDa, for example about 3000 kDa, about 4000 kDa, about 5000 kDa, about 6000 kDa, about 7000 kDa, about 8000 kDa, or about 9000 kDa.
Preferably, the high molecular weight of hyaluronic acid or salt thereof (HMWHA) of plant origin (called Geenluronic®) of the present invention: a. may comprise a disaccharide ADi-HA generated by chondroitinase AC enzymatic hydrolysis of the Tremella extract, b. does not contain (absence of) chondroitin 4 and 6 mono-sulfates, c. may further have a content of glucuronic acid comprised in a range from 80% to 98%, preferably from 85% to 95%, for example 90%, d. may further have an average molecular weight comprised in a range from about 1500 kDa to about 20000 kDa, preferably from about 1650 kDa to about 15000kDa, more preferably from about 2000 kDa to about 10000 kDa, for example about 3000 kDa, about 4000 kDa, about 5000 kDa, about 6000 kDa, about 7000 kDa, about 8000 kDa, or about 9000 kDa.
The Applicant has also performed a dose-response and time-course study of Greenluronic® on CaCo-2 cells. Before studying the permeability and transport of Greenluronic®, the human immortalized colorectal adenocarcinoma (CaCo-2) cell line was used to perform a dose-response study to exclude any cytotoxic effects. The analysis was performed comparing the effects of Greenluronic® to sodium hyaluronate testing them at the same concentration (ranging from 0.125 to 1 pg/pL) on cell viability and ROS production in CaCo-2 cells in a timecourse study (from 2 h to 6 h). The cell viability of the CaCo-2 cells, measured by 3-(4, 5-D imethy lthiazol-2-y l)-2,5- diphenyltetrazolium bromide (MTT) assay, showed time and concentration dependent effects of both substances (Figure 3A), and the beneficial effects compared to control (p<0.05) were maintained during all periods of stimulation excluding any cytotoxic effect at all dosage tested. In particular, the cells treated with Greenluronic® 1 pig/pil showed high viabilities compared to control (p<0.05) and compared to other concentrations tested (p<0.05) suggesting that Greenluronic® 1 pig/pil is non-toxic to intestinal epithelial cells exhibiting the best profile also compared to sodium hyaluronate at the same concentration and time (p<0.05). Additional experiments were carried out in order to confirm the safety of Greenluronic® on intestinal epithelium analyzing if the substances tested could induce oxidative stress. For this reason, ROS production was evaluated on CaCo-2 cells from 2h to 6h of stimulations with both Greenluronic® and sodium hyaluronate. As shown in Figure 3B, none of the concentrations tested was able to increase the ROS production maintaining them at normal physiological conditions. Greenluronic® 1 pig/pil maintains low ROS level during all periods analyzed better than the other concentrations tested and then all sodium hyaluronate concentration, and it was maintained for all further experiments.
Further, the Applicant has also carried out a permeability analysis of Greenluronic® using an in vitro model of intestinal barrier. To assess permeability, to obtain additional information about the Greenluronic® intestinal absorption, further experiments were carried out performing a 3D in vitro model in order to mimic the in vivo complexity of the intestinal barrier. In this context, 1 pig/pil Greenluronic® and 1 pig/pil sodium hyaluronate were tested from 2h to 6h in order to measure transepithelial electrical resistance (TEER) values, the apparent permeability coefficient (Papp) values and the HA concentration to predict their bioavailability. The data obtained show that the intestinal adsorption has a physiological trend as can be observed from the analysis of TEER and tight junction (TJ). In particular, the passage through the intestinal epithelium demonstrates that both sodium hyaluronate and Greenluronic® were able to maintain the epithelial integrity increasing the ionic flux of the paracellular exchanges across the intestinal epithelial compared to control (p<0.0001). Indeed, Greenluronic® demonstrates the better effect compared to sodium hyaluronate during all time of the stimulation (p<0.0001 ) as reported in Figure 4A. Afterwards, also the evaluation of TJ confirmed these results; indeed Greenluronic® exerted the greatest effects on occludin (p= 0.0286, about 31%, Figure 4B), claudin-1 (p= 0.0299, about 37%, Figure 4C) and zonula occludens-1 (ZO-1) (p= 0.0299, about 50%, Figure 4D) compared to sodium hyaluronate and compared to control value (reported as 0 line, p<0.05). From these encouraging results, which confirmed the correct functioning of the intestinal epithelium, further experiments were carried out measuring the permeability rate, analyzing the flux of non-electrolyte tracers (expressed as permeability coefficient as reported) and how much HA has crossed the intestinal barrier to reach the target site. Data obtained from the analysis of basolateral environment (Figure 4E) confirmed our previous findings since the amount of Greenluronic® was higher compared to sodium hyaluronate (p<0.05) with a maximum effect at 4h compared to sodium hyaluronate (about 20%, p<0.013). In addition, the data obtained from the quantification of the basolateral level (Figure 4F) supported the hypothesis about the importance of predicting human absorption; Greenluronic® has a higher amount of HA that crosses the barrier and reaches the plasma level compared to control (p<0.0001) and compared to sodium hyaluronate (about 30%, p<0.0001) with the greatest effects between 4h and 5h.
The effects of Greenluronic® crossed intestinal barrier on chondrocytes have been tested. Since the exogenous hyaluronic acid into articular cartilage has a direct biological effect on chondrocytes several experiments were carried out to explore the effect of Greenluronic®, compared to sodium hyaluronate on chondrocytes after intestinal absorption in terms of mitochondrial metabolism and cell proliferation. As expected (Figure 5A), both 1 pig/pil Greenluronic® and sodium hyaluronate were able to improve cell viability compared to control (p<0.05); in particular Greenluronic® induces the main effect on cell viability (about 50%, p<0.05) compared to sodium hyaluronate reducing the ROS production (about 38% p<0.05), as reported in Figure 5B. Furthermore, as reported in figure 5C, Greenluronic® induces an improvement in cell proliferation compared to control (p<0.05) and compared to sodium hyaluronate about 60% indicating that Greenluronic® is able to stimulate the proliferative activity of chondrocytes. Since the importance of the activity on cell proliferation includes the ability to modulate joint producing HA in cells, the HA quantification in chondrocytes (Figure 5D) revealed that a large amount of HA present in Greenluronic® and sodium hyaluronate were captured by chondrocytes after intestinal passage compared to the control (p<0.05). In particular, approximately 75% of HA was induced by Greenluronic® compared to sodium hyaluronate (p<0.05) in chondrocytes confirming that HMWHA is better utilized by chondrocytes.
The effects of HA crossed intestinal barrier on chondrocytes under OA condition have been tested. From the data obtained under physiological conditions, it can be assumed that Greenluronic® is also effective after oral administration and is an important starting point for determining the success of therapy in joint damage, such as OA. Oxidative stress and inflammation are known to be involved in cartilage degeneration of OA and it is similarly approved that the degree of anti-inflammatory, immunomodulatory, analgesic and anti-OA effects of HA is determined by MW and route of administration. Based on these results, in the last phase, the in vitro study was conducted by analyzing the effects of both 1 g /pl of Greenluronic® and sodium hyaluronate on T/C-28a2 cells pretreated with 10pg/ml of lipopolysaccharide (LPS) for 24 hours in order to simulate the condition of OA. The effects of chondrocyte metabolism were shown in Figure 6 where the beneficial effects of Greenluronic® can be observed. Specifically, chondrocytes treated only with 10pg/ml of LPS significantly reduced cell viability (panel 6A, about 10%) and improved ROS production (panel 6B about 23%) compared to control (p<0.05) but this effect was significantly reduced by the presence of both agents. In particular, Greenluronic® was able to counteract these negative effects caused by LPS alone (p<0.05) better than sodium hyaluronate (about 47% on cell viability and 2 time on ROS production respectively, p<0.05). These data were also confirmed by nuclear factor kappa B (NFkB) analysis (Figure 60) in which the beneficial potential of Greenluronic® against inflammation, a key point in the mechanisms involved during OA processes, was observed. Indeed, the cells treated with only 10pg/ml of LPS increased inflammatory processes compared to control (about 18%, p<0.05) assuming the beginning of chronic processes leading to cell death, and this situation was reversed following stimulation with both HA agents.
In particular, 1 pig/pil Greenluronic® was able to reduce the negative effect produced by LPS (about 1.25 times more) better than sodium hyaluronate (about 50%, p<0.05). This recovery mechanism was confirmed also by proliferation assay (panel 6D) in which T/C-28a2 cells lost their proliferative properties when treated only with 1 Opig/ml of LPS (p<0.05 compared to control).
On the contrary, both 1 pig/pil Greenluronic® and sodium hyaluronate counteract this negative effect compared to control (p<0.05) but the Greenluronic® was able to restore the damage about 62% compared to sodium hyaluronate (p<0.05), supporting its use during OA injuries. Finally, this recovery mechanism was also confirmed by the analysis of HA which showed that under OA conditions Greenluronic® is able to improve a much higher amount of HA released in stressed chondrocytes than sodium hyaluronate at the same concentration, approximately about 21 % (p<0.05).
In order to demonstrate that LPS is able to reproduce the OA condition in vitro leading to chondrocyte death, additional experiments were carried out to explore the involvement of the apoptosis process. As reported in Figure 7, several markers related to apoptotic processes were evaluated in response to 10pig/ml of LPS and to both 1 pig/ml Greenluronic® and sodium hyaluronate. In particular, the stimulation with 10pig/ml of LPS treatment on T/C-28a2 cells enhanced BAX and Caspase 9 activities (Figure 7A and 7B), about 22% and 18% compared to control (p<0.05), indicating a dramatic improvement of the apoptosis process supporting the chondrocyte death during OA. Contemporary, the stimulation with both 1 pig/pil Greenluronic® and sodium hyaluronate added after 10pig/ml of LPS caused a statistically significant reduction of both these markers. In particular, Greenluronic® exerted the main effects compared to sodium hyaluronate on Bax (about 2 time less, p<0.05) and Caspase-9 activities (about 1.5 time less, p<0.05) suggesting that Greenluronic® contributes to cell protection. These data were also confirmed by the activation of risk pathways such as mitogen-activated protein kinases/extracellular signal-regulated kinase (ERK/MAPK) activity (Figure 7C) which demonstrated that Greenluronic® reverts the 10|jg/ml LPS damage, activating the survival pathways and restoring the chondrocyte to normal conditions (about 25% compared to sodium hyaluronate, p<0.05).
Finally, to explore the possible effector molecules responsible for the maintenance of chondrocytes wellbeing, the activity of cyclin D1 , osteoprotegerin (OPG) and CD44 were investigated. As reported in Figure 8 (panel A to D), 1 Opig/ml of LPS confirmed its negative effect on T/C-28a2 cells compared to control (p<0.05) downregulating OPG activity, CD44 and cyclin D1 expressions (about 18%, 8% and 12% compared to control, respectively) modifying negatively chondrocytes activity. Conversely, both 1 pig/pil Greenluronic® and sodium hyaluronate were able to reduce the damage induced by 10 pig/ml of LPS (p<0.05), confirming the positive role of HA contained in two agents in stimulating chondrocytes metabolism. In particular, 1 pig/ml Greenluronic® appears to be able to induce main effects compared to sodium hyaluronate (p<0.05) to counteract the negative effects of OA inductor. Indeed, 1 pig/ml Greenluronic® is able to restore the damage induced by 10pig/ml of LPS in all parameters tested (about 60% for OPG, one time more for CD44 and 57% for cyclin D1 expression, p<0.05), suggesting that it could ameliorate chondrocyte pathological conditions by activating them through the markers responsible for articular joint homeostasis.
DISCUSSION
Current guidelines for the treatment of OA suggest many conventional approaches to improve this chronic condition. For example, pharmacological treatment, which is characterized by NSAIDs [Magni, A., Agostoni, P., Bonezzi, C. et al. Management of Osteoarthritis: Expert Opinion on NSAIDs. Pain Ther 10, 783-808 (2021). https://doi.org/10.1007], opioids, and cyclooxygenase (COX)-2-specific drugs, is an accepted method considered only as a "palliative” method since it reduces the symptoms but does not address the essential problem of cartilage degeneration [25], In addition, conventional therapies can cause possible side effects especially for long periods of use, which can reduce the compliance for the onset of gastrointestinal, cardiovascular, and other adverse effects [26], Furthermore, the conventional therapies often use HA injections to treat knee OA and to improve the functions of the knee joint, naming this method viscosupplementation [Migliore A, Procopio S. Effectiveness and utility of hyaluronic acid in osteoarthritis. Clin Cases Miner Bone Metab. 2015 Jan-Apr; 12(1 ):31 -3. doi: 10.11138/ccmbm/2015.12.1.031.]. It was reported that intra-articular HA improves synovial fluid elasticity and viscosity, by decreasing the release of pain mediators and proinflammatories from synovial cells [27], Otherwise, a recent systematic review, based on the analysis of pain relief and functional improvement, concluded that the routine use of HA injections does not produce so many benefits for the patient with no clinical relevance, because of the pain caused [28], According to the current protocol, HA should be administered repeatedly into the joint cavity but this multiple injections caused must discomfort associated with the injections in patients and increase the risk of complication by the repetition of injections [Toshiyuki Tashiro, Satoshi Seine, Toshihide Sato, Ryosuke Matsuoka, Yasunobu Masuda, Naoshi Fukui, "Oral Administration of Polymer Hyaluronic Acid Alleviates Symptoms of Knee Osteoarthritis: A Double-Blind, Placebo-Controlled Study over a 12-Month Period", The Scientific World Journal, vol. 2012, Article ID 167928, 8 pages, 2012. https://doi.org/10.1100/2012/167928]. Notwithstanding, HA is the useful tool in the management of patients with OA, since clinical data indicate its ability to reduce pain and improve joint function, with a potential ability to modify chondrocytes activity [29], It should also be taken into account that the administration of HA by intraarticular injection can also cause adverse effects such as infectious arthritis and cartilage damage [Zhang Y, Chen X, Tong Y, Luo J, Bi Q. Development and Prospect of Intra-Articular Injection in the Treatment of Osteoarthritis: A Review. J Pain Res. 2020 Aug 4;13:1941-1955. doi: 10.2147/JPR.S260878.]. Therefore, the possibility of administering HA orally represents a considerable advantage [Fallacara A, Baldini E, Manfredini S, Vertuani S. Hyaluronic Acid in the Third Millennium. Polymers (Basel). 2018 Jun 25;10(7):701. doi: 10.3390/polym10070701 .]. From this point of view several studies have explored new approaches for consistent and pain-free administration of HA, reporting positive effects after oral administration and suggesting that it may have beneficial therapeutic effects on patients with OA [30-32],
Advantageously, it is possible to design a new nutraceutical based on plant-derived HA, called Greenluronic® which is able to counteract the harmful consequences of OA.
The chemical analysis carried out by the Applicant, has revealed that Greenluronic® contains a large amount of HA with a chemical profile useful to be a new nutraceutical product.
In addition, the presence of a high molecular weight ingredient related to HA supports its use to counteract the adverse effects of OA, since high molecular weight HA is nowadays the best treatment option for knee OA by intraarticular injection [Altman RD, Bedi A, Karlsson J, Sancheti P, Schemitsch E. Product Differences in Intraarticular Hyaluronic Acids for Osteoarthritis of the Knee. Am J Sports Med. 2016 Aug;44(8):2158-65. doi: 10.1177/0363546515609599.]. However, as reported in literature, elevated levels of HA in serum after its oral administration in vivo model was also reported. Indeed, therapeutic efficacy of HA against lameness was found to be greater with oral than intraarticularly because this way of administration dissipates out of the joint the main amount of HA within 14-18 h; HA diffuses out tissues via the bloodstream, circulating throughout the body, and is rapidly eliminated [Gupta RC, Lail R, Srivastava A, Sinha A. Hyaluronic Acid: Molecular Mechanisms and Therapeutic Trajectory. Front Vet Sci. 2019 Jun 25;6:192. doi: 10.3389/fvets.2019.00192], In addition, several study in human, revealed that the patients with OA need to be must visit clinics repeatedly and must undergo the discomfort associated with the injections undergoing also to an increase if complications associated to the repetition of injections [Toshiyuki Tashiro, Satoshi Seine, Toshihide Sato, Ryosuke Matsuoka, Yasunobu Ma-suda, Naoshi Fukui, "Oral Administration of Polymer Hyaluronic Acid Alleviates Symptoms of Knee Osteoarthritis: A DoubleBlind, Placebo-Controlled Study over a 12-Month Period", The Scientific World Journal, vol. 2012, Article ID 167928, 8 pages, 2012. https://doi.org/10.1100/2012/167928]. Considering these overt and potential disadvantages, it is far more desirable use HA by oral administration to ameliorate OA condition. Indeed, some studies have suggested that the symptoms of knee OA might indeed be alleviated by HA ingestion and other studies report also positive effects of orally administered HA on improving joint functionality in mild to moderate knee osteoarthritis [Toshiyuki Tashiro, Satoshi Seine, Toshihide Sato, Ryosuke Matsuoka, Yasunobu Masuda, Naoshi Fukui, "Oral Administration of Polymer Hyaluronic Acid Alleviates Symptoms of Knee Osteoarthritis: A DoubleBlind, Placebo-Controlled Study over a 12-Month Period", The Scientific World Journal, vol. 2012, Article ID 167928, 8 pages, 2012. https://doi.org/10.1100/2012/167928; H. Iwaso and T. Sato, "Examination of the efficacy and safety of oral administration of Hyabest J, highly pure hyaluronic acid, for knee joint pain,” Journal of Japanese Society of Clinical Sports Medicine, vol. 17, no. 3, pp. 566-572, 2009; I. Nagaoka, K. Nabeshima, S. Mura-kami et al., "Evaluation of the effects of a supplementary diet containing chicken comb extract on symptoms and cartilage metabolism in patients with knee osteoarthritis,” Experimental and Therapeutic Medicine, vol. 1 , no. 5, pp. 817— 827, 2010; Bogdan OA, Cerbu S. Oral hyaluronic acid in patients with knee osteoarthritis. Progress in Nutri-tion 2019; Vol. 21, N. 1 : 243-245 DOI: 10.23751/pn.v21i1 .8185], In addition, the inter-national evidence-based guidelines agree that knee OA management requires both non-pharmacological, and pharmacological approaches and suggest to initiate a background therapy with chronic symptomatic slow-acting drugs for OA such as HA by oral administration [Guadagna S, Barattini DF, Pricop M, Rosu Serba. Oral hyaluronan for the treatment of knee osteoarthritis: a systematic review. Progress in Nutrition 2018; Vol. 20, N. 1 : 537-544 DOI: 10.23751/pn.v20i4.7581], Consequently, the effects of Greenluronic® were analyzed mimicking the human oral ministration in vitro, since orally administered HA should be absorbed and distributed to the knee joints where it exerts its biological activities. The results obtained from the 3D model that mimics intestinal absorption clearly demonstrated that oral administration is possible. Bioavailability experiments indicated that orally administered HA is effectively absorbed and biodistributed to the chondrocytes and exerts its biological functions in those tissues. Advantageously, Greenluronic® has a higher amount of HA that reaches the plasma level compared to control (p<0.05) and compared to sodium hyaluronate within 4h and 5h, confirming the hypothesis that Greenluronic® improves the absorption during physiological time of intestinal digestion and improving its bioavailability. In addition, Greenluronic® treatment indicated that a substantial part of HA is absorbed without damaging the intestinal epithelium; this is a crucial point since HA has a role in decreasing the permeability by enhancing the tight junction proteins. In epithelial cells, the formation of tight junctions plays an important role in the intestinal barrier, and this is mediated by proteins such as claudins, occludin, and ZO-1 that are necessary for epithelial barrier activity [33], These proteins are critical in maintaining homeostatic intestinal permeability in multiple intestinal inflammatory diseases, supporting a gut-joint axis in OA pathogenesis and progression [Guido G, Ausenda G, lascone V, Chisari E. Gut permeability and osteoarthritis, towards a mechanistic understanding of the pathogenesis: a systematic review. Ann Med. 2021 Dec;53(1):2380-2390. doi: 10.1080/07853890], In particular, dysbiosis-related gut permeability determined lower mRNA levels of TJ, ZO-1 and occludin, and higher LPS plasma levels in vivo model which has a positive association of synovial LPS with inflammation and disease severity in articular chondrocytes in OA patients [Huang ZYu, Chen J, Li BLei, et al.. Faecal microbiota transplantation from metabolically compromised human donors accelerates osteoarthritis in mice. Ann Rheum Dis. 2020;79(5):646- 656; Kolasinski SL, et al.. 2019 American college of rheumatology/arthritis foundation guideline for the management of osteoarthritis of the hand, hip, and knee. Arthritis Rheum. 2020;72(2):220— 233]. For this reason, several nutraceuticals evaluate the intestinal integrity as first step to ameliorate pain and disease progression of OA [Guido G, Ausenda G, lascone V, Chisari E. Gut permeability and osteoarthritis, towards a mechanistic understanding of the pathogenesis: a systematic review. Ann Med. 2021 Dec;53(1):2380-2390. doi: 10.1080/07853890], using al-so HA as a multifunctional agent [Kotla NG, Bonam SR, Rasala S, Wankar J, Bohara RA, Bayry J, Rochev Y, Pandit A. Recent advances and prospects of hyaluronan as a multifunctional therapeutic system. J Control Release. 2021 Aug 10;336:598-620. doi: 10.1016/j.jconrel.2021.07.002]; in particular, Kotla et al. demonstrated that the treatment with HA upregulated the expression of the tight junction proteins claudin and occludin [34], These three proteins are pivotal because ZO-1 connects claudin and occludins to the cytoskeleton so they are indicators of good gut barrier functions [35], Furthermore, it has been demonstrated that Greenluronic® is able to maintain epithelial integrity and the ionic exchanges across the intestinal barrier suggesting that this proteoglycan is able to pass the cell monolayer without negatively altering the epithelium. Subsequently, the second important purpose of this work was to test the ability of Greenlronic® to stimulate chondrocyte biological activity under physiological and pathological conditions. As expected, Greenluronic® was able to stimulate cell viability and induce chondrocyte proliferation without causing adverse effects, also compared to conventional HA supplementation. Indeed, the beneficial effects of Greenluronic® due to the presence of HMWHA into chondrocytes activity supporting the hypothesis of this use on joint-inflammatory conditions. Since OA is a disease of the whole joint and a multifactorial entity, there are various therapeutic strategies that involve numerous fields of medicine: rheumatology, orthopedics, geriatrics, psychiatry, general practitioners and physiotherapists. The goal of OA therapy is to reduce pain and increase patients' quality of life. For this purpose, HA has shown not only beneficial effects on articular cartilage trophism, but also antinociceptive effects with a significant reduction in pain [Jimbo S, Terashima Y, Teramoto A, Takebayashi T, Ogon I, Watanabe K, Sato T, Ichise N, Tohse N, Yamashita T. Antinociceptive effects of hyaluronic acid on monoiodoacetate-induced ankle osteoarthritis in rats. J Pain Res. 2019 Jan 3; 12: 191-200. doi: 10.2147/JPR.S186413], In particular, the beneficial effects of Greenluronic® has also been confirmed by the quantity of HA, contained in this new formulation, which reached the target site and was absorbed on the joint without damaging it. Consequently, therapeutic effects of Greenluronic® on OA conditions may necessarily require the improved absorption of HA. Moreover, we pre-treated T/C-28a2 cells with 10pig/ml of LPS to mimic the osteoarthritic phenotype as reported by Zhang et al. [36], Our data supports the literature [37, 38] showing that HMWHA may bind to CD44 on chondrocytes to exert its biological activities, demonstrating that the association of HA with CD44 increased the HA absorption/production suppressing proinflammatory processes. The binding of HA, present in Greenluronic®, to CD44 also suppresses the expression of the apoptosis process, which again contributes to the damage of chondrocytes and improve its activity, regulating cartilage production, improving OPG activity, and proliferation process, modulating cyclin D1 expression. Taken together, these results suggest that Greenluronic® is the best choice to maintain chondrocyte behaviour during the inflammatory condition related to OA condition, and therefore, the application of this innovative HA form could be an excellent strategy to restore OA damage.
EXPERIMENTAL PART
Materials and Methods
1 . Agents preparation
Greenluronic® is obtained from White Tremella (Silver Ear), which is a traditional foodstuff with medicinal application in China [39], The production process involves several steps necessary to obtain a final extract and includes a new technology based on patent N°WO2021/250566 and N°WC2020/245809 from Vivatis Pharma GmbH, Gruner Deich 1-3, 20097 Hamburg, Germany.
Briefly, the process involves steps of extraction, purification, refining by alcohol solution, sieving and crushing. The resulting powder is then packed and tested for metals and stored [40, 41], In addition, the sodium hyaluronate (Merck Life Science, Rome, Italy) was tested to verify the mechanism of action of Greenluronic®. All these substances are prepared directly in water for HA determination or directly in Dulbecco's Modified Eagle's Medium (DMEM, Merck Life Science, Rome, Italy) without phenol red and supplemented with 0.5% foetal bovine serum (FBS, Merck Life Science, Rome, Italy), 2 mM L-glutamine (Merck Life Science, Rome, Italy) and 1 % penicillinstreptomycin (Merck Life Science, Rome, Italy) for biological analysis.
2. HPLC analysis
The determination of the HA was also confirmed by HPLC (Shimadzu, Kyoto, Japan) analysis according to the method reported in literature [42], as described in Appendix A (Supplementary Material 1 and 2). Briefly; 20 pL of TRIS buffer (3.0 g TRIZMA base, 4.0 g sodium acetate trihydrate, 1.46 g sodium chloride, and 50 mg crystalline bovine serum albumin dissolved in 100 mL of 0.12 M HCI, pH 7.3 with 6 M HCI. All chemicals are purchased from Merck Life Science, Rome, Italy), 30 pL of chondroitinase AC solution (Merck Life Science, Rome, Italy) (diluted to 10U/mL with water), and 20 pL of Greenluronic® test solution (200 mg dissolved in 100 mL of water) were pipetted into a conical 1.5 mL vial. The vial was placed in a warm water bath at 37°C for 3 h. After cooling at room temperature, the sample was diluted to 1 mL by adding 930 pL of mobile phase A (reagent purchased from Merck Life Science, Rome, Italy and column from Phenomenex srl, Bologna, Italy) (see supporting information in Appendix A) and the mixture was analyzed by HPLC-UV and HPLC-HRMS systems. A control solution was prepared by replacing the enzyme aliquot with TRIS buffer. 3. Colorimetric Determination of Hyaluronic Acid
The assay performed to quantify the concentration of HA on material samples was the same reported in literature [43], Briefly, 1 mg of raw sample was dissolved in 1 mL of deionized water and 200pil of resuspended samples were displaced in new eppendorf, diluted with 1.2 mL of sulfuric acid (Merck Life Science, Rome, Italy) with 0.0125M tetraborate (Merck Life Science, Rome, Italy), shaked for 20 seconds and then boiled at 100°C for 5 min. Once the samples were allowed to cool on ice, 20pil of 0.15% hydroxydiphenyl (Merck Life Science, Rome, Italy) (dissolved in 0.5% NaOH, Merck Life Science, Rome, Italy) was added and stirred; 10Opil of each sample was placed in a 96 multi-well plate and the absorbance was measured at 340 nm by a spectrophotometer (Infinite 200 Pro MPlex, Tecan, Mannedorf, Switzerland). The data obtained were compared to a calibration curve generated using glucuronic acid (0, 0.25, 0.5, 1 , 1.5, 2 mg/ml Merck Life Science, Rome, Italy) [44] and the results were expressed as mean (%w/w) ± SD compared to control (0 line).
4. Molecular weight determination
The determination of the molecular weight of HA before exploring its biological effects, was carried out using 1% agarose gel, following the method reported in literature [45], Briefly, 0.3 g agarose (Merck Life Science, Rome, Italy) was dissolved in 30ml of Tris-acetate-EDTA (TAE) buffer (48.5g tris base, 11.4 mL acetic acid and 0.5 M EDTA pH 8, all substances were purchased from Merck Life Science, Rome, Italy) and the solution was heated for 30 seconds in a microwave at high power. The gel was poured into the holder and allowed to solidify before performing a pre-run at 100V for 45 min, using the Mini-Sub Cell GT System (Bio-Rad, Hercules, California, USA). In the meantime, samples were prepared dissolving 200pig of raw samples in 16 l of TAE buffer 1x. Before running the gel, 4 l of loading buffer (0.2% Bromophenol Blue, 1 ml of TAE 1x and 8.5ml of glycerol which were purchased from Merck Life Science, Rome, Italy) were added to each sample and to the molecular weights (mixture of 5 l of Select-HA HILadder and 5 l Select-HA Mega Ladder, Echelon Biosciences, Tebu-Bio srl, Magenta, Italy). The samples were run at 100V until the samples reached 1cm from the end of the gel. Then, the gel was hydrated in H2O for 24h at room temperature in agitation and then the gel was placed in 30% ethanol with 0.015% Stains All dye (Merck Life Science, Rome, Italy) for 24 h in the dark. The gel was decoloured for 30 min in H2O in the dark before proceeding with image acquisition using ChemiDoc™ Touch Imaging System (Bio-Rad, Hercules, California, USA). The image obtained was analyzed by Image Lab 3.0 software (Bio-Rad Hercules, California, USA).
5. Cell culture
The human epithelial intestinal cells, CaCo-2, purchased from the American Type Culture Collection (ATCC), were cultured in Dulbecco's Modified Eagle's Medi-um/Nutrient F-12 Ham (DMEM-F12, Merck Life Science, Rome, Italy) containing 10% FBS (Merck Life Science, Rome, Italy), 2 mM L-glutamine and 1% penicillin- streptomycin (Merck Life Science, Rome, Italy) maintaining in an incubator at 37°C and 5% CO2 [33], The cells used for the experiments were at passage numbers between 26 to 32 in order to preserve the integrative of paracellular permeability and transport properties [46] maintaining the similarity to the intestinal absorption mechanism following oral intake in humans. The cells were plated in different manner to perform several experiments including 1 x104 cells in 96 well plates to study cell viability by MTT-based In Vitro Toxicology Assay Kit (Merck Life Science, Rome, Italy) and ROS production using cytochrome C (Merck Life Science, Rome, Italy) in a complete medium. 8h before the stimulation the cells were incubated with DMEM without red phenol and supplemented with 0.5% FBS (Merck Life Science, Rome, Italy), 2 mM L-glutamine and 1 % penicillin-streptomycin (both from Merck Life Science, Rome, Italy) at 37 °C to synchronize them. In addition, 2x104 cells were plated on 6.5 mm Transwell® (Corning® Costar®, Merck Life Science, Rome, Italy) with 0.4 pm pore polycarbonate membrane insert (Corning® Costar®, Merck Life Science, Rome, Italy) in a 24 well plate to perform the absorption analyses [47], Cells plated on Transwell® insert were maintained in complete medium which was changed every other day on the basolateral and apical sides for 21 days before the simulations [48], Before to the stimulation, on the apical side the medium was brought to pH 6.5 as the pH in the lumen of the small intestine, while the pH 7.4 on the basolateral side represented blood [49, 50], This in vitro model is widely used [47, 51] and accepted by European Medicines Agency (EMA) and FDA to predict absorption, metabolism and bioavailability of several substances after oral intake in humans [52, 53],
The immortalized human juvenile costal chondrocyte cell line T/C-28a2 (purchased from Merck Life Science, Rome, Italy) was cultured in DMEM-F12 medium supplemented with 10% FBS (Merck Life Science, Rome, Italy), 2mM L- glutamine (Merck Life Science, Rome, Italy), and antibiotics (50 Ul/ml penicillin and 50 pg/ml streptomycin, Merck Life Science, Rome, Italy)) and maintained in an incubator at 5% CO2 and 95% humidity [54], This cell line is representative and the most commonly used cells for mimicking joints [55] and they were used between passages 3 and 10 [56], For the experiments 1 xi o4 cells were seeded in 96 well plates to study cell viability by MTT-based In Vitro Toxicology Assay Kit (Merck Life Science, Rome, Italy), ROS pro-duction using cytochrome C (Merck Life Science, Rome, Italy) and Crystal Violet (Merck Life Science, Rome, Italy) in a complete medium; additionally, 1 x106 cells were plated on a 6-well to determine HA concentration, using quantification kit, and to analyze molecular pathways by Western blot analysis or ELISA kit.
6. Experimental protocol
In order to analyze the beneficial effects of hyaluronic acid on articular joints in humans after oral intake, the experiments were divided in two steps; the aim in the first one was to verify the ability of HA to cross the intestinal barrier in vitro model excluding negative effects and in the second one was to check the direct effects on chondrocytes analyzing several parameters and mechanism of actions. For this reason, in intestinal CaCo-2 cells a dose-response study ranging from 0.125 to 1 pg/pL [57] was performed to assess the concentration able to exert beneficial effects on cell viability and ROS production. Subsequently, the best concentration of Greenluronic® and hyaluronic acid salt were tested on intestinal in vitro barrier model, to verify intestinal integrity through TEER measurement, tight junction analysis by ELISA kit and permeability assay by Papp measurement also analyzing the total amount of hyaluronic acid that has crossed the intestinal barrier. For all these experiments, cells were treated in a time-dependent manner from 2 h to 6 h as reported in the literature [33], In addition, after each stimulation the basolateral medium was collected to be used on chondrocytes cells. T/C-28a2, a chondrocyte cell line widely used to study articular joints, were treated for 3 days [58] and, at the end of stimulation, the mitochondrial metabolism, cell proliferation, ROS production and hyaluronic acid quantification were tested. Finally, in order to mimic OA conditions, further experiments were performed pre-treating T/C-28a2 with 1 Opig/ml of LPS (Merck Life Science, Rome, Italy) for 24h [36] and then stimulating with Greenluronic® and sodium hyaluronate for 3 days to evaluate if they are able to restore the damage. In these conditions, the survival mechanisms and articular recovery were investigated.
7. Cell viability
The analysis of cell viability was performed using a classical technique based on MTT-based In Vitro Toxicology Assay Kit (Merck Life Science, Rome, Italy) [59], following the manufacturer's instructions. Indeed, at the end of stimulation, the cells were incubated with 1 % MTT dye for 2 h in an incubator at 37°C, 5% CO2, and 95% humidity, and then the purple formazan crystals were dissolved in equal volume of MTT Solubilization Solution. The absorbance was analyzed by spectrophotometer (In-finite 200 Pro MPlex, Tecan, Mannedorf, Switzerland) at 570 nm with correction at 690 nm, and results were expressed compared to the control (0% line) which represented untreated cells. The results reported an increase in the percentage of viable cells compared to the control and indicate a higher number of viable cells plus the control. This strategy can lead to a high level of safety of the stimulation and consequently to a correct analysis of the results.
8. In Vitro Intestinal Barrier Model
An intestinal barrier model, using CaCo-2 cells, was performed to analyze the passage through the intestinal barrier of Greenluronic® and sodium hyaluronate, having as final destination the chondrocyte where they could exert their beneficial effects. For this reason, the TEER values were determined with EVOM3, coupled with STX2 chopstick electrodes (World Precision Instruments, Sarasota, FL, USA); this assay was carried out every 2 days for 21 days until reaching a TEER value > to 400 Qcm2 before the stimulation [33, 60], time required for the cell monolayer formation, for cell differentiation and for the exposition of the intestinal villi. On day 21, the medium at the apical and basolateral environments were changed to create different pH conditions: pH around 6.5 at the apical level (acidic pH mimicking lumen of small intestine) and pH around 7.4 at the basolateral level (neutral pH mimicking human blood) [48], The cells were kept for 15 min at 37°C and 5% CO2, after that the TEER values were measured again before the start of the experiment to verify the stabilization of the values. The cells were stimulated with Greenluronic® and sodium hyaluronate for 2 h to 6 h before the successive analysis, including the permeability assay measured by Papp analysis [33], Briefly, the Papp (cm/s) was calculated with the following formula [33, 48]:
Papp = dQ/dt -»i 1/m0 -»i 1/A -»i V Donor dQ: amount of substance transported (nmol or pg); dt: incubation time (sec); mO: amount of substrate applied to donor compartment (nmol or pg);
A: surface area of Transwell membrane (cm2); VDonor: volume of the donor compartment (cm3).
Negative controls without cells were tested to exclude Transwell membranes influence.
9. Occludin Quantification Assay
The Human Occludin ELISA kit (OCLN kit, MyBiosource, San Diego, CA, USA) analyzed the occludin presence in CaCo-2 cell lysates, according to the manufacturer's instruction [33], Briefly, CaCo-2 cells were lysed with cold Phosphate Buffered Saline (PBS, Merck Life Science, Rome, Italy) 1 x, centrifuged at 1500 g for 10 min at 4 °C and 100 pL of each sample was transferred to the strip well before the incubation at 37 °C for 90 min. The supernatants were removed, and the strips were incubated with 100 pL of Detection Solution A for 45 min at 37 °C; then, the strips were washed with Wash Solution and incubated with 100 pL of Detection Solution B for additional 45 min. At the end of this time, 90 pL of Substrate Solution was added followed by an incubation for 20 min at 37 °C in the dark, and then 50 pL of Stop Solution was used to block the enzymatic reaction. The plate was analyzed by a spectrophotometer (Infinite 200 Pro MPlex, Tecan, Mannedorf, Switzerland) at 450 nm. The concentration is expressed as pg/mL compared to a standard curve (range from 0 to 1500 pg/ml) and the results are expressed as percentage (%) versus control (0 line).
10. Claudin 1 Detection
The Human Claudin 1 was measured in CaCo-2 lysates by ELISA kit (Cusabio Technology LLC, Huston, USA), following the manufacturer's instructions [33], Briefly, the cells were lysed with cold PBS 1 x (Merck Life Science, Rome, Italy) and centrifuged at 1500* g for 10 min at 4 °C. 100 pL of each sample was added to ELISA plate and incubated at 37 °C for 2h; then, the plate was washed and 100 pL of Biotin-antibody were added to the wells and incubated for 1 h at 37 °C. After this time the wells were washed and 100 pL of HRP-avidin were added in each well and the samples were incubated for 1 h at 37 °C. 90 pL of TMB Substrate was also added to the samples and the plate was incubated for 20 min at 37 °C protected from light. At the end, 50 pL of Stop Solution was used to stop the reaction and the plate was analyzed by a spectrophotometer (Infinite 200 Pro MPlex, Tecan, Mannedorf, Switzerland) at 450 nm. The concentration is expressed as pg/mL comparing data to the standard curve (range from 0 to 1000 pg/ml) and the results are expressed as percentage (%) versus control (0 line).
11 . ZO-1 detection
The Human Tight Junction Protein 1 ELISA kit (MyBiosource, San Diego, CA, USA) was measured in CaCo-2, following the manufacturer's instructions [33], Briefly, the cells were rinsed with ice-cold PBS 1x (Merck Life Science, Rome, Italy) and processed with two freeze-thaw cycles; then cell lysates were centrifuged for 5 minutes at 5000xg at 4°C. 10Opil of each sample were collected and incubated on the ELISA plate at 37 °C for 90 min; after washing, 100 pL of Detection Solution A were added to each well and incubated for 45 min at 37 °C. The wells were washed and 100 pL Detection Solution B was added to the samples. After an incubation of 45 min, the wells were washed again and 90 pL of Substrate Solution were added in each well and then the samples were incubated for 20 min at 37 °C in the dark. Finally, 50 pL of Stop Solution were added and then the plates were read by a spectrophotometer (Infinite 200 Pro MPlex, Tecan, Mannedorf, Switzerland) at 450 nm. The concentration is expressed as pg/mL comparing data to standard curve (range from 0 to 1000 pg/ml) and the results are expressed as percentage (%) versus control (0 line).
12. Crystal violet staining
At the end of stimulation time, the cells were fixed with 1 % glutaraldehyde (Merck Life Science, Rome, Italy) for 15 min at room temperature, washed, stained with 10Opil 0.1 % aqueous crystal violet (Merck Life Science, Rome, Italy) for 20 min at room temperature and solubilized with 10Opil 10% acetic acid before reading the absorbance at 595 nm using a spectrophotometer (Infinite 200 Pro MPlex, Tecan, Mannedorf, Switzerland). The estimated number was determined comparing data to the control cells normalized to TO (measurement at the beginning of the stimulation) [61], The results are expressed as percentage (%) versus control (0 line).
13. ROS production
The quantification of superoxide anion release was obtained following a standard protocol based on the reduction of cytochrome C [61], and the absorbance in culture supernatants was measured at 550 nm using the spectrophotometer (Infinite 200 Pro MPlex, Tecan, Mannedorf, Switzerland). The 02 rate was expressed as the mean ± SD (%) of nanomoles per reduced cytochrome C per microgram of protein compared to the control (0 line) [61].
14. Quantification of hyaluronic acid in cell culture
At the end of stimulations, both cell types were lysed with 100 pi of cold PBS1x to measure the total HA following the instructions of Hyaluronic Acid ELISA Kit (Clue-Clone). Briefly, 50 pil of sample and reagent A were added in each well and after gently shaking the plate was incubated for 1 h at 37°C. At the end, the wells were washed three times and 100 pil of reagent B were added before incubating the plate for 30 minutes at 37°C, then 90 pil of substrate solution were added before incubating the plate for 20 minutes at 37°C. At the end, 50 pil of stop solution were added immediately before reading at 450 nm by spectrophotometer (Infinite 200 Pro MPlex, Tecan, Mannedorf, Switzerland) [62, 63], The results are expressed as means ± SD (%) versus control (0 line).
15. ERK/MAPK activity
The analysis of ERK/MAPK activity was performed using the InstantOneTM ELISA (Thermo Fisher, Milan, Italy) on chondrocytes lysates [64], Briefly, 50 pL of lysate samples prepared in Lysis Buffer were tested in ELISA microplate strips after the incubation for 1 h at room temperature in a microplate shaker pre-coated with the antibody cocktail. After that, the strips were incubated with the detection reagent for 20 min before stopping the reaction with stop solution. The absorbance was measured by a spectrophotometer at 450 nm (Infinite 200 Pro MPlex, Tecan, Mannedorf, Switzerland) and the results expressed as means ± SD (%) versus control (0 line). 16. OPG activity
The OPG/TNFRSF11 B Duo Set (R&D Systems, Minneapolis, MN, USA) was applied according to the manufacturer's instructions to verify the OPG involvement [65], Briefly, 100 pL of samples or standards were added to the well and incubated for 2h at room temperature protected from light and, after washing, 100 pL of the Detection Antibody were added to each well and incubated as previously described. After 2h, 100 pL of the working dilution of Streptavidin-HRP A were added to each well and incu-bated for 20 minutes at room temperature. At the end of the time, 100 pL of Substrate Solution were added to each well, incubated for 20 minutes at room temperature and then 50pL of Stop Solution were used to stop the enzymatic reaction. The absorbance of each well was measured at 450 nm by a spectrophotometer (Infinite 200 Pro MPlex, Tecan, Mannedorf, Switzerland) and the results were interpolated with the standard curve (6.25 to 625pg/ml) and the results were expressed as means ± SD (%) compared to control (0 line).
17. NFKB analysis
The NF-kB (p65) Transcriptional factor Assay kit was carried out to analyze the NF-KB DNA binding activity, following the manufacturer's instruction (Cayman Chemical Company, Ann Arbor, Ml, USA) [66], The concentration was calculated by comparing results to the standard curve generated by NF-kB (p65) Transcriptional factor positive control (ranging from 0 to lOpil/well according to different scaled dilution) and re-ported as means ± SD (%) compared to control (0 line).
18. BAX assay
BAX activity was determined in chondrocytes lysates using ELISA kit (Human Bax ELISA Kit, MyBiosource, San Diego, CA, USA) according to the manufacturer's instruction [67], The absorbance of the samples was measured at 450 nm by a spectrophotometer (Infinite 200 Pro MPlex, Tecan, Mannedorf, Switzerland) and the results were compared to the standard curve (range from 0 to 2000 pg/ml) and expressed as means ± SD (%) normalized to control value (0 line).
19. Caspase 9 assay
The Caspase 9 activity was investigated in chondrocytes lysates by ELISA kit (Caspase 9 Human ELISA Kit.Thermoscientific, Waltham, Massachusetts, USA), according to the manufacturer's instructions, reading the sample's absorbance at 450 nm with a spectrometer (Infinite 200 Pro MPlex, Tecan, Mannedorf, Switzerland). The data were obtained by comparing to a standard curve (ranging from 1.6 to 100 ng/ml) and the results are expressed as means ± SD (%) compared to control value (0 line) [68],
20. Western blot analysis
At the end of each stimulation, chondrocytes were washed with ice-cold PBS 1X (Merck Life Science, Rome, Italy), and lysed using Complete Tablet Buffer (Roche, Ba-sel, Switzerland) supplemented with 2mM sodium orthovanadate (Na3VO4), 1 mM phenylmethanesulfonylfluoride (PMSF) (Merck Life Science, Rome, Italy), 1 :50 mix Phosphatase Inhibitor Cocktail (Merck Life Science, Rome, Italy), and 1 :200 mix Protease Inhibitor Cocktail (Merck Life Science, Rome, Italy) to obtain a total protein extract which was centrifuged at 14.000 g for 20 min at 4 °C. 35pig of proteins for each extract were resolved on 8% and 10% SDS-PAGE gel and transferred to a polyvinylidene difluoride (PVDF) membrane which was incubated overnight with the specific primary antibodies such as Cyclin D1 (1 :500, Santa Cruz, CA, United States) and CD44 (1 :500, Santa Cruz, CA, United States). All protein expressions were normalized and verified through p-actin detection (1 :5000, Merck Life Science, Rome, Italy), and ex-pressed as mean ± SD (%) compared to control value (0 line).
21. Statistical analysis
Data obtained from each experimental protocol and assay were collected and analyzed using GraphPad Prism 7 statistical software through mixed variance analysis. In particular, for all growth curves, bar graphs and line graphs, five independent experiments performed in triplicates and included for statistical analysis were conducted. All time points in growth curves were presented as the mean of the three biological replicates with mean errors <5%. The two-tailed Student's t-test followed by Welch's t test to analyze two groups Multiple comparisons between groups were analyzed by two-way ANOVA followed by a two-tailed Dunnett post hoc test. Error bars in the bar charts and line charts represent the standard deviation. For TEER analyses, one-way ANOVA followed by Bonferroni post hoc tests was performed to see if the means were significantly different between groups. All results were expressed as mean ± SD of at least 5 independent experiments performed in triplicates. Differences with a p value < 0.05 were considered statistically significant. Data normality was assessed with the Kolmogorov-Smirnov test.
Conclusions
As demonstrated by these findings, the results of our study performed by the Applicant of the present invention show that this new form of plant HA is likely absorbed and distributed to the chondrocytes, while preserving its biological activities. Although the in vitro data are very clear and promising, in vivo or even in human studies would be needed to confirm these observations, before assuming an absolute effectiveness of this HA from plant derived. Thus, despite the fact that our data derived from an in vitro study and therefore need further validation, the results of the present study about the effectiveness in improving chondrocytes function in conditions that mimic OA, may support the hypothesis that the oral administration of Greenluronic® in humans can be considered a valid therapeutic strategy to obtain beneficial therapeutic effects during OA. In particular, it can be hypothesized that these promising beneficial effects are relevant not only to the joint but to any OA-induced damage.
Preferred embodiments of the present invention FRn are as follows.
FR1 . A mixture that comprises or, alternatively, consists of:
- a high molecular weight of hyaluronic acid or salt thereof (HMWHA) of plant origin present that comprises or, alternatively, consists of disaccharide ADi-HA obtained by chondroitinase AC enzymatic hydrolysis of the Tremella extract. FR2. The mixture according to FR1 , wherein the high molecular weight of hyaluronic acid or salt thereof (HMWHA) of plant origin does not contain chondroitin 4 and 6 mono-sulfates.
FR3. The mixture according to FR1 o Fr2, wherein the high molecular weight of hyaluronic acid or salt thereof (HMWHA) of plant origin has a content of glucuronic acid comprised in a range from 80% to 98%, preferably from 85% to 95%, for example 90%.
FR4. The mixture according to anyone of FR1-3, wherein the high molecular weight of hyaluronic acid or salt thereof (HMWHA) of plant origin has an average molecular weight comprised in a range from about 1500 kDa to about 20000 kDa, preferably from about 1650 kDa to about 15000kDa, more preferably from about 2000 kDa to about 10000 kDa, for example about 3000 kDa, about 4000 kDa, about 5000 kDa, about 6000 kDa, about 7000 kDa, about 8000 kDa, or about 9000 kDa.
FR5. The mixture according to anyone of FR1-4, wherein said high molecular weight of hyaluronic acid or salt thereof (HMWHA) of plant origin has a higher amount of HA that crosses the barrier and reaches the plasma level compared to control (p<0.0001) and compared to sodium hyaluronate (about 30%, p<0.0001) with the greatest effects between 4h and 5h.
FR6. The mixture according to anyone of FR1-5, wherein said high molecular weight of hyaluronic acid or salt thereof (HMWHA) of plant origin in an amount of 1 pig/pil is was able to reduce the negative effect produced by LPS (about 1.25 times more) better than sodium hyaluronate (about 50%, p<0.05).
FR7. The mixture according to anyone of FR1-6, wherein said mixture is for use:
- (I) in a method for maintaining joint homeostasis and preventing the harmful processes of osteoarthritis, and/or
- (II) in a method for preventing and restoring the degradation of cartilage, and/or
- (ill) in a method for the treatment of osteoarthritis OA.
FR8. A composition comprising a mixture according to anyone of FR1-7, and optionally at least one excipients or carrier of pharma or food grade, wherein said composition is for use:
- (I) in a method for maintaining joint homeostasis and preventing the harmful processes of osteoarthritis, and/or
- (ii) in a method for preventing and restoring the degradation of cartilage, and/or
- (ill) in a method for the treatment of osteoarthritis OA.
FR9. The composition according to FR8, wherein said composition is preferably a food or nutraceutical composition, more preferably for oral use.
FR10. A process for preparing the mixture according to anyone of FR1-7, wherein said process comprises steps of extraction of the high molecular weight of hyaluronic acid or salt thereof (HMWHA) from White Tremella (Silver Ear), purification, refining by alcohol solution, sieving and crushing to provide a powder of HMWHA. Abbreviation list
ADAMTS = disintegrin and metalloproteinase with thrombospondin motifs
ANOVA = one-way analysis of variance
CaCo-2 = the human immortalized colorectal adenocarcinoma cell line
CD44 = differentiation cluster 44
COX-2 = cyclooxygenase 2
DMEM/F12 = Dulbecco's modified Eagle's medium/Nutrient F-12 Ham
EFSA = European Food Safety Authority
ELISA = Enzyme-Linked Immunosorbent Assay
EMA = European Medicines Agency
ERK = extracellular signal-regulated kinases
ERK/MAPK = mitogen-activated protein kinases/extracellular signal-regulated kinase
FBS = fetal bovine serum
FBS= foetal bovine serum
FDA = US Food and Drug Administration
GAGs = glycosaminoglycan heteropolysaccharides family
HA = hyaluronic acid
HMWHA = high-molecular weight HA
HPLC = High Performance Liquid Chromatography
HRMS = high-resolution mass spectrometry
IBD = inflammatory bowel disease
IL-10 = interleukin (IL) -10
LMWHA = low molecular weight HA
LPS = lipopolysaccharide
MTT = 3-(4,5-Dimethy I thiazol-2-y l)-2,5-diphenyl tetrazolium bromide
MMPs = matrix metalloproteinases
MPK-1 = mitogen-activated protein kinase phosphatase 1
MTT = 3-(4,5-Dimethylthiazol-2-yl)-2,5-Diphenyltetrazolium Bromide
Na3VO4 = sodium orthovanadate
NFkB = nuclear factor kappa B
NSAIDs = non-steroidal anti-inflammatory drugs
NO = nitric oxide (NO)
OA = Osteoarthritis
OPG = osteoprotegerin
Papp = apparent permeability coefficient
PBS = phosphate buffered saline
PGE2 = prostaglandin E2 PMSF = phenylmethanesulfonyl fluoride PVDF = polyvinylidene difluoride
RHAMM = hyaluronan mediated motility receptors ROS = reactive oxygen species TEER = transepithelial electrical resistance T/C-28a2 = human chondrocyte cells TJ = tight junction
ZO-1 = zonula occludens-1
Appendix A
Supplementary Materials 1: HPLC-UV method
• Column: Phenomenex Synergi Polar 4 pm 150 x 4.6mm preceded by a Security guard Polar and kept at room temperature
• Mobile phase A: 340 mg of tetrabutylammonium bisulphate dissolved in 1000 mL of water HPLC grade.
• Mobile phase B: 340 mg of tetrabutylammonium bisulphate dissolved in 330 mL of water HPLC grade, then after the solution is at room temperature, brought to 1000 mL with acetonitrile.
• Wavelength: 240 nm
• Volume of injection: 30 pl
• Flow rate: 1.1 mL/min
• Gradient elution program:
Time (min) Mobile phase B%
0.00 20
7.00 65
12.00 65
12.50 20
22.50 20
Table 2
Supplementary Materials 2: HPLC-HRMS method
• Thermo Scientific Q-Exactive plus
• Column: Phenomenex Synergi Polar 4 pm 150 x 2.0 mm preceded by a Security guard Polar and kept at room temperature
• Mobile phase A: 0.1 % formic acid in water
• Mobile phase B: 0.1 % formic acid in acetonitrile
• Volume of injection: 5 pl
• Flow rate: 0.200 mL/min
• Gradient elution program: Time (min) Mobile phase B%
0.00 15
4.00 50
9.50 50
10.00 15
15 15
Table 3
• Positive full scan.
NMR SPECTRUM
To understand the molecular underpinnings of structural and functional properties of polysaccharides, nuclear magnetic resonance (NMR) spectroscopy is a powerful tool for this type of study, e.g.13C NMR measurements can reveal the structural and dynamical features of polysaccharides. Thus, isotopic enrichment with 13C is essential to apply advanced NMR experiments since the NMR-active isotopes, including 13C, are naturally very low (1.1 % and 0.4%). https://doi.org/10.1016/bs.abr.2019.11.005. Indeed, as reported by Rampratap et al., efficient isotope enrichment strategies for hyaluronic acid and other polysaccharides promise to facilitate characterization by NMR in this important and growing field of research, spanning biology, pharmaceuticals and other (industrial) applications doi: 10.1016/j.carbpol.2023.121063. As reported in the figure below, the 13C spectrum was recorded with a technique called CPMAS (to increase the sensitivity) which is inherently not quantitative. In this technique, carbons directly bonded to protons (e.g. CH3) will be overestimated while other carbons (e.g. COOH) will be underestimated. The experimental time to record the spectrum was 14 hours. As reported in Figure 11, the natural abundance of 13C is only 1.1 %. In particular, the numbers are the chemical shifts in ppm associated with the 13C resonances. One should be able to identify the expected compounds from these chemical shift values. The analysis of Greenluronic® revealed the same structure as hyaluronic acid.
The results obtained are similar to what was observed in other data present in the literature, as reported in Figure 12.
The chemical composition of hyaluronic acid is shown in table 4.
Figure imgf000031_0001
Table 4
Data Interpretation: As reported by Ret et al. [https://doi.Org/10.1016/j.carbpol.2018.10.003], a very effective, reliable, and widespread method for the determination of the degree of substitution (DS) of polysaccharides is 1 H
NMR spectroscopy. The area below the signals in 1 H NMR spectra is proportional to the number of protons responsible for the signal. It can obtain quantitative information about the material's chemical structure. DS of modified HA is usually calculated from the ratio of signal integrals of methyl protons of the N-acetyl residue of HA as backbone reference peak and specific signals of protons of the grafted moiety. Low-molecular-weight HA derivatives can be easily characterized by NMR spectroscopy due to the very low viscosity of the solutions. Still, the accuracy of DS determination by conventional 1D 1 H NMR is strongly limited in highly viscous environments, where interactions between polymer chains are very strong, and aggregation phenomena may occur. This leads to low-resolution spectra with broad signals and reduced signal intensity and to the uncertainty of up to 15% in the value of DS obtained from integrating the peaks. A standard approach to overcome the problem of high viscosity of polyelectrolytes such as HA, chitosan or alginates is a decrease of molecular weight. HA degradation by enzymatic or acid hydrolysis was reported as a suitable method for a higher resolution in NMR analysis. Drawbacks of these methods are the time-consuming sample preparation and the risk of a loss of pH-sensitive grafted moieties. Thus, a direct and correct analysis of high molecular weight HA (derivatives) would be advantageous NMR signals of specific groups are quantitative only when mobile, i.e., not implied in an ordered structure such as helical structures, in which hydrogen bonds can stabilize in stereoregular polymers or by specific interactions. In the case of high molecular weight HA (such as Greeniuronic®) spectra acquired at room temperature in D2O, proton mobility of acetyl groups, backbone protons and anomeric protons varies between 41% and 56.4%. Increasing the temperature resulted in an increase of proton mobility to 74-83%, but it was observed that interactions between different chains were still present at 85 °C. In all cases, the protons of the N-acetyl groups were more affected than those of the sugar units. It was concluded that high molecular weight HA 1 H NMR spectroscopy could not be used to test the acetyl content in the HA structure.
It is well known that the viscosity of solutions of polyelectrolytes such as HA can be reduced by increasing the ionic strength of the solution because the repulsion between negatively charged disaccharide units decreases and the polymer changes from a rod-like-structure toward the flexible conformation of the random coil. This should lead to a higher mobility of the macromolecules and a higher proton mobility.
Therefore, the graph in Figure 13 (from Ret et al.) shows the same NMR spectrum as Greeniuronic: Greeniuronic® ppm 4.37 and HA ppm from bibliography 4.79. This slight difference in proton shift is due to the high molecular weight of Greeniuronic® (1650kDa) compared to HA from the bibliography, which has a molecular weight of 1200kDa.
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Claims

1 . A mixture that comprises or, alternatively, consists of:
- a high molecular weight of hyaluronic acid or salt thereof (HMWHA) of plant origin present that comprises or, alternatively, consist of disaccharide ADi-HA obtained by chondroitinase AC enzymatic hydrolysis of the Tremella extract, wherein the high molecular weight of hyaluronic acid or salt thereof (HMWHA) of plant origin does not contain chondroitin 4 and 6 mono-sulfates.
2. The mixture according to claim 1 , wherein the high molecular weight of hyaluronic acid or salt thereof (HMWHA) of plant origin has a content of glucuronic acid comprised in a range from 80% to 98%, preferably from 85% to 95%, for example 90%.
3. The mixture according to anyone of claims 1 to 2, wherein the high molecular weight of hyaluronic acid or salt thereof (HMWHA) of plant origin has an average molecular weight comprised in a range from about 1500 kDa to about 20000 kDa, preferably from about 1650 kDa to about 15000kDa, more preferably from about 2000 kDa to about 10000 kDa, for example about 3000 kDa, about 4000 kDa, about 5000 kDa, about 6000 kDa, about 7000 kDa, about 8000 kDa, or about 9000 kDa.
4. The mixture according to anyone of claims 1 to 3, wherein said high molecular weight of hyaluronic acid or salt thereof (HMWHA) of plant origin has a higher amount of HA that crosses the barrier and reaches the plasma level compared to control (p<0.0001) and compared to sodium hyaluronate (about 30%, p<0.0001) with the greatest effects between 4h and 5h.
5. The mixture according to anyone of claims 1 to 4, wherein said high molecular weight of hyaluronic acid or salt thereof (HMWHA) of plant origin in an amount of 1 pig/pil was able to reduce the negative effect produced by LPS (about 1.25 times more) better than sodium hyaluronate (about 50%, p<0.05).
6. The mixture according to anyone of claims 1 to 5, wherein said mixture is for use:
- (I) in a method for maintaining joint homeostasis and preventing the harmful processes of osteoarthritis, and/or
- (II) in a method for preventing and restoring the degradation of cartilage, and/or
- (ill) in a method for the treatment of osteoarthritis OA.
7. A composition comprising a mixture according to anyone of claims 1 to 6, and optionally at least one excipients or carrier of pharma or food grade, wherein said composition is for use:
- (I) in a method for maintaining joint homeostasis and preventing the harmful processes of osteoarthritis, and/or
- (ii) in a method for preventing and restoring the degradation of cartilage, and/or
- (ill) in a method for the treatment of osteoarthritis OA.
8. The composition according to claim 7, wherein said composition is preferably a food or nutraceutical composition, more preferably for oral use.
9. A process for preparing the mixture according to anyone of claims 1-6, wherein said process comprises steps of extraction of the high molecular weight of hyaluronic acid or salt thereof (HMWHA) from White Tremella (Silver Ear), purification, refining by alcohol solution, sieving and crushing to provide a powder of HMWHA.
PCT/IB2023/057469 2022-07-21 2023-07-21 A new high molecular weight of hyaluronic acid or salt thereof of plant origin for use in maintaining joint homeostasis and preventing the harmful processes of osteoarthritis. WO2024018437A1 (en)

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