Hyaluronic acid-lysine copper chelate and its application

Abstract

This invention provides a hyaluronic acid-lysine-copper chelate with oil-controlling, repairing, and hair-growth-promoting effects, and its applications in cosmetics and pharmaceuticals. The chelate of this invention is characterized by a copper content of 1%–9% (mass percentage), a lysine to copper ion molar ratio of 1:0.4–0.6, and a hyaluronic acid disaccharide unit to lysine molar ratio of 1:0.1–2. This chelate can be used to prepare cosmetics and medical products for improving skin and preventing hair loss.

Description

Technical Field

[0001] This invention relates to a hyaluronic acid-lysine-copper chelate with oil-controlling, repairing and hair-growth-promoting effects, and the application of said chelate in the cosmetic and pharmaceutical fields. Background Technology

[0002] Minerals are a class of inorganic nutrients and are essential elements for the human body, enabling the skin to maintain optimal health. Mineral ions play multiple roles in the skin, including intercellular communication, catalysis of biological reactions, and promotion of electron transfer. Metal ions play a significant role in skin tissue in maintaining skin barrier homeostasis, resisting external environmental stress, anti-inflammatory and soothing effects, and improving aging. In human skin, sodium, chloride, potassium, magnesium, and calcium are present in high concentrations, distributed from the stratum corneum to the dermis; iron, copper, and zinc are present in relatively low concentrations. Copper, as one of the important trace elements with a content of less than 0.01% in the human body, can cause weakness and skin ulcers if it is deficient [Reference: Hu Wenyu. Trace elements in cosmetics, Guangdong Journal of Trace Elements Science, 1999, 6(10): 13-15].

[0003] Copper ions can regulate the activity of antioxidant enzymes such as superoxide dismutase and glutathione peroxidase, as well as small molecule antioxidants such as glutathione in the skin's endogenous antioxidant system, and scavenge free radicals in the body; assist vitamin C oxidation and the synthesis of elastin on the skin, upregulate the level of TGF-β1, and stimulate the synthesis of extracellular matrix proteins by activating lysine oxidase. In synergy with calcium ions, they enhance the fiber strength at the dermal papilla / epidermal junction and improve facial aging; downregulate the sharp increase of pro-inflammatory cytokines such as IL-6 and IL-8 after skin injury, inhibit inflammation, and alleviate heat damage [Reference: Ma Xiaoyu, Yan Xiaojuan, Bao Xijun. Skin biological effects of metal mineral ions and their application in the cosmetic field. Daily Chemical Industry (Chinese and English), 2023, 53(11):1315-1324].

[0004] In addition, reports have disclosed that copper can regulate tyrosinase activity, regulate melanocytes to produce melanin to maintain the skin's natural color; it can produce disulfide cross-links in the stratum corneum, which can promote hair growth; copper ions have an inhibitory effect on both type I and type II 5α-reductase, the latter of which is associated with androgen-dependent diseases such as benign prostatic hyperplasia, acne vulgaris, hirsutism in women, seborrheic dermatitis, male pattern baldness, and pseudohermaphroditism [Reference: Yasuro Sugimoto, et al. Cations Inhibit Specifically Type I 5α-Reductase Found in Human Skin. Invest Dermatol. 1995,104(5):775-778].

[0005] Hyaluronic acid (HA), also known as hyaluronic acid, is composed of (1- β -4) D -glucuronic acid and (1- β -3) N -acetyl- Dα-Glucosamine disaccharide units linked together in a chain-like polyanionic polysaccharide are one of the main components of the skin extracellular matrix. In skin tissue, it plays a role in water retention, maintaining extracellular space, regulating the flow and concentration of extracellular cations, regulating the differentiation and migration of epidermal keratinocytes, promoting normal epidermal cell differentiation, and scavenging free radicals. WO00 / 08061 discloses the reaction product of hyaluronic acid and natural amino acids and its application in cosmetic and pharmaceutical compositions.Hyaluronic acid and lysine can be combined through two mechanisms: salt formation and amide bonding. The combination is mainly used as a moisturizer in cosmetics. In the medical field, it can be used to promote wound healing and recovery from bedsores, repair damaged nasal mucosa and corneal epithelial cells in dry eye, and regenerate bone and cartilage. [References: ① Giuseppe Alonci, et al. Physico-Chemical Characterization and In Vitro Biological Evaluation of a BionicHydrogel Based on Hyaluronic Acid and L-Lysine for Medical Applications. Pharmaceutics, 2021, 13, 1194; ② Giorgio Felzani, et al. Effect of LysineHyaluronate on the Healing of Decubitus Ulcers in Rehabilitation Patients. Adv Ther, (2011) 28(5):439-445; ③ Adriana Di Benedetto, et al. Osteogenic and Chondrogenic Potential of the Supramolecular Aggregate T-LysYal®. Frontiers in Endocrinology, 2020, 11, 285. Class III medical device products prepared by combining HA with multiple amino acids, including lysine, can be used to treat and prevent skin wrinkles [F. Svolacchia, et al. Evaluation of the efficacy and safety of hyaluronic acid and supplemented with amino acids, and glutathione or colin, for the prevention and treatment of wrinkles on the face, neck, décolleté and hands. European Review for Medical and Pharmacological Sciences, 2023, 27 (3 Suppl): 99-108]. In recent years, various copper-containing compounds have been used to prepare skincare products with effects such as skin repair, soothing, anti-wrinkle, and firming. However, existing inorganic copper salts can disrupt the emulsification systems commonly used in cosmetics, and small-molecule organic copper compounds are lost from the skin surface relatively quickly.

[0006] This invention provides an organic macromolecular copper chelate. The combination of lysine copper chelate and hyaluronic acid is beneficial to the stability of cosmetic formulation systems, has high moisturizing properties and better skin adhesion, and has stronger efficacy. It can be widely used in the preparation of cosmetics and medical products with oil control, repair and hair growth promotion effects. Summary of the Invention

[0007] The purpose of this invention is to provide a hyaluronic acid-lysine-copper chelate with oil-controlling, repairing and hair-growth-promoting effects, as well as the application of this chelate in cosmetics and pharmaceuticals for improving skin and preventing hair loss.

[0008] The hyaluronic acid-lysine-copper chelate of the present invention is characterized in that: ① The copper ion content is 1%~9% (g / g); ② The molar ratio of lysine to copper ions is 1:0.4~0.6; The molar ratio of hyaluronic acid disaccharide units and lysine is 1:0.1~2.

[0009] The hyaluronic acid-lysine-copper chelate of the present invention is further characterized in that the lysine-copper chelate is combined with hyaluronic acid in the form of a salt, and is represented by general formula (1). (1) Where X, Y, and Z represent the number of hyaluronic acid disaccharide units, X>Y>Z.

[0010] The hyaluronic acid-lysine-copper chelate of the present invention is further characterized in that the lysine-copper chelate is combined with hyaluronic acid in an amide manner, represented by general formula (2). (2) Where X, Y, and Z represent the number of hyaluronic acid disaccharide units, X>Y>Z.

[0011] The hyaluronic acid-lysine-copper chelate of the present invention is further characterized in that the copper ion content is 5%~7% (g / g). The hyaluronic acid-lysine-copper chelate of the present invention, as described in claim 1, is further characterized in that the molar ratio of the hyaluronic acid disaccharide units and lysine is 1:0.2~1.5, preferably 1:0.8~1.2.

[0012] The hyaluronic acid-lysine copper chelate of the present invention is characterized in that the average molecular weight of the hyaluronic acid bound to the lysine copper chelate refers to the average molecular weight determined by high performance size exclusion chromatography-multiangle laser scattering (GPC-MALLS), which is in the range of 5 kDa to 2000 kDa, preferably in the range of 10 kDa to 800 kDa, and more preferably in the range of 200 kDa to 500 kDa.

[0013] The hyaluronic acid-lysine-copper chelate with the technical features of this invention, in addition to having high moisturizing properties and better skin adhesion, has a significant inhibitory effect on 5α-reductase, can effectively control oil, prevent and treat acne vulgaris, improve the skin's water-oil balance, prevent seborrheic alopecia and improve hair quality, repair skin and mucous membrane damage, promote the production of elastin and collagen, prevent and reduce skin wrinkles, make the skin smooth and elastic, play an anti-wrinkle and firming role, and improve facial aging.

[0014] This invention relates to a hyaluronic acid-lysine-copper chelate that can be used to prepare topical skin products with oil-controlling, repairing, and hair growth-promoting effects, including cosmetics and medical products. Cosmetics include skin care products and bath products, while medical products include medical devices and pharmaceuticals for the prevention and treatment of acne vulgaris, seborrheic alopecia, bedsores, skin and mucous membrane injuries, and facial and neck wrinkles.

[0015] The hyaluronic acid-lysine-copper chelate of this invention can be added to commonly used cosmetic ingredients to formulate various forms such as solutions, gels, ointments, creams, or lotions. These can be used to prepare daily bath products, such as shower gels, shampoos, conditioners, facial cleansers, hair creams, shaving creams, shaving foams, and shaving lotions. They can also be used to prepare daily skincare products, such as balancing moisturizing lotions, balancing moisturizing sprays, balancing moisturizing masks, oil-controlling toners, lotions, softening lotions, exfoliating gels, acne-reducing gels, balancing moisturizing creams, face creams, body creams, hand creams, foot creams, repairing essences, moisturizing creams, repairing lotions, moisturizing lipsticks, lip glosses, lip stains, hair dyes, hair growth tonics, and scalp care solutions.

[0016] The hyaluronic acid-lysine-copper chelate of this invention can be added to commonly used excipients in the pharmaceutical field to formulate various dosage forms such as solutions, gels, and creams. These can be used to prepare topical skin products, such as acne creams, hair growth lotions, acne gels, and hair growth tonics. They can also be used to prepare repair materials for skin and mucous membranes, such as wound dressings, pressure ulcer pads, scar patches, dental and nasal rinsing solutions, gynecological washes, and hyaluronic acid injections. Attached Figure Description

[0017] Figure 1Infrared spectrum of sodium hyaluronate (hyaluronic acid). 3385 cm⁻¹ represents the stretching vibration of OH, and 2895 cm⁻¹ represents the stretching vibration of CH. Figure 2 Infrared spectra of hyaluronic acid-lysine copper chelates (samples 1-3 in Example 1 of this invention). The IR spectra of hyaluronic acid-lysine copper chelates bound by salt formation show characteristic peaks similar to Lys-Cu-Lys.

[0018] Figure 3 Infrared spectra of hyaluronic acid-lysine copper chelates (samples 1-9 in Example 1 of this invention). The IR spectra of hyaluronic acid-lysine copper chelates bonded by amide bonds show that the stretching vibration band of OH is broadened and extended, shifting to approximately 3420 cm⁻¹, while the stretching vibration of CH is shifted to approximately 2929 cm⁻¹.

[0019] Figure 4 Superimposed infrared spectra of hyaluronic acid-lysine copper chelate and sodium hyaluronate, bound by both salt formation and amide bonds. The IR spectrum of the hyaluronic acid-lysine copper chelate bound by salt formation shows that the stretching vibrations of OH and CH are at 3445 cm⁻¹, 3263 cm⁻¹, 3045 cm⁻¹, and 2946 cm⁻¹, respectively; the asymmetric C=O stretching vibrations are at 1661 cm⁻¹ and 1585 cm⁻¹, exhibiting characteristic peaks similar to Lys-Cu-Lys; the fingerprint region at 613 cm⁻¹ still shows the characteristic HA peak. The IR spectrum of the hyaluronic acid-lysine copper chelate bound by amide bonds shows that the stretching vibration band of OH in hyaluronic acid is broadened and extended, with the 3385 cm⁻¹ peak shifting to approximately 3420 cm⁻¹ at higher wavenumbers; the CH stretching vibration peak at 2895 cm⁻¹ shifts to approximately 2929 cm⁻¹ at higher wavenumbers; the fingerprint region still shows the characteristic HA peak.

[0020] Figure 5 Comparison of the relative inhibition rates of hyaluronic acid-lysine copper chelate, copper sulfate, and lysine-hydrochloride copper chelate against 5α-reductase. Using 0.2 mM dutasteride (DTS) solution as a positive control, all three test samples showed significant inhibitory effects on 5α-reductase. The inhibitory effect of the hyaluronic acid-lysine copper chelate of this invention was better than that of copper sulfate and lysine-hydrochloride copper chelate.

[0021] Figure 6Comparison of skin adhesion of lysine hydrochloride copper chelate, copper hyaluronic acid, and hyaluronic acid-lysine copper chelate: a. Loss of copper ions in the skin test area (ug); b. Cumulative loss of copper ions in the skin test area (%). After 12 wipings, the cumulative loss of copper ions was measured. The results were 95.68% for lysine hydrochloride copper chelate, 86.68% for copper hyaluronic acid, and 71.50% for hyaluronic acid-lysine copper chelate. The skin adhesion of the hyaluronic acid-lysine copper chelate of this invention is better than that of lysine hydrochloride copper chelate and copper hyaluronic acid. Detailed Implementation

[0022] The following examples are provided to better illustrate the present invention and are not intended to limit the invention.

[0023] Example 1 Hyaluronic acid-lysine-copper chelate Sodium hyaluronate and lysine copper chelate were mixed and dissolved evenly. An appropriate amount of ethanol was added for precipitation and purification. After filtration, the mixture was dehydrated with anhydrous ethanol and dried under reduced pressure to obtain a blue granular powder, which is the hyaluronate lysine copper chelate obtained by salt formation in this invention (samples 1-1 to 1-5).

[0024] Sodium hyaluronate and lysine copper chelate were mixed and dissolved evenly. Catalysts N-(3-dimethylaminopropyl)-N'-ethylcarbodiimide hydrochloride (EDCI) and N-hydroxysuccinimide (NHS) were added. After the reaction was kept at a certain temperature, an appropriate amount of ethanol was added for precipitation and purification. After filtration, the mixture was dehydrated with anhydrous ethanol and dried under reduced pressure to obtain a blue granular powder, which is the hyaluronic acid lysine copper chelate obtained by the present invention through amide bond bonding (samples 1-6 to 1-10).

[0025] In the above preparation process, by adjusting the ratio of sodium hyaluronate and lysine hydrochloride copper chelate, the amount of catalysts EDCI and NHS, and the pH value of the reaction solution, samples with different copper ion contents can be obtained, as shown in the table below.

[0026] Preparation of the comparative compound copper hyaluronic acid: Sodium hyaluronic acid and copper chloride were mixed and dissolved evenly, and then an appropriate amount of ethanol was added for precipitation and purification. After filtration, the mixture was dehydrated with anhydrous ethanol and dried under reduced pressure to obtain the final product.

[0027]

[0028] Copper ions [Cu] 2+ Method for determining the content (%(g / g)): Take an appropriate amount of sample, weigh it accurately, dissolve it completely in water, and prepare a solution with a concentration of about 1%. Add 2 to 3 drops of 1-(2-pyridinium azo)-2-naphthol (PAN) indicator, and titrate with 0.01 mol / L disodium ethylenediaminetetraacetate (EDTA-2Na) standard solution. The titration endpoint is when the solution color changes from dark blue to light green. Correct the titration result with a blank test.

[0029] Cu in the sample 2+ Content is expressed as a percentage by mass and is calculated using the following formula:

[0030] In the formula: V0: The volume of EDTA-2Na standard titrant consumed in titrating the blank solution, in ml; V1: The volume of EDTA-2Na standard titrant consumed in titrating the sample solution, in ml; c: Concentration of EDTA-2Na standard titrant, mol / L; m: Mass of the sample, in grams; h (%): Loss on drying of the test sample.

[0031] Method for determining lysine [Lys%(g / g)] content: Citrate buffer (0.2 mol / L, pH 5.5): Weigh 3.84 g of citric acid and 5.16 g of trisodium citrate, dissolve them separately in 100 mL of distilled water, take 15.21 g of citric acid solution and 93 g of trisodium citrate solution, mix well, and the solution is ready.

[0032] Ninhydrin reagent: Weigh 1g of ninhydrin and dissolve it in 25mL of 95% ethanol. Separately weigh 40mg of tin dichloride and dissolve it in 25mL of citrate buffer. Mix the two solutions, shake well, and store in a brown bottle in a refrigerator for later use.

[0033] Lysine standard solution: Accurately weigh 50 mg of lysine standard, dilute with distilled water to a final volume of 1000 ml, and obtain a standard solution with a concentration of 50 μg / ml.

[0034] Test solution: Weigh about 0.1 g (m1) of this product accurately, dissolve it in water to 100 mL, then take 7.0 g (m2) (7.0 mL based on a solution density of 1.000 g / mL), dilute it in water to 50 mL, and the solution is ready.

[0035] Standard curve: Take 0, 0.2, 0.4, 0.6, 0.8, and 1.0 mL of lysine standard solution, add water to a final volume of 1.0 mL, add 2 mL of ninhydrin reagent to each solution, mix well, incubate at 80℃ for 30 min, cool to room temperature in a water bath, dilute with water to a final volume of 10 mL, mix well, and use tube 0 as a blank. Measure the absorbance at 570 nm. Plot a standard curve with lysine concentration on the x-axis and absorbance on the y-axis, and perform linear regression.

[0036] Test sample determination: Take 1.0 mL of the test sample solution, add 2 mL of ninhydrin reagent, mix well, incubate at 80℃ for 30 min, cool to room temperature in a water bath, then dilute with water to 10 mL, mix well, and measure the absorbance at 570 nm. Calculate using the following formula.

[0037] In the formula: Ci is the concentration of lysine obtained from the regression equation, in μg / ml; m1 is the mass of the test sample, in grams; m2 is the mass of the diluted sample solution, in grams; h (%) represents the loss on drying of the test sample.

[0038] According to the literature (Bitter T, Muir HM. A modified carbazole reaction l JJ. Anal Biochem. 1962. 4: 330-333.), the glucuronic acid content T% (g / g) of the sample was determined by the modified carbazole method.

[0039] Chloride ions [Cl] - Method for determining the content (%(g / g)): Accurately weigh approximately 0.1 g of the sample and place it in a burette. Add 50 ml of ultrapure water to dissolve it. Titrate with silver nitrate titrant (0.1 mol / L) using potentiometric titration. Correct the titration result with a blank experiment. Each 1 ml of silver nitrate titrant (0.1 mol / L) is equivalent to 3.55 mg of chloride ions.

[0040] The molar ratio of copper ions to lysine [Cu] 2+ [Lys (mol / mol)] is calculated using the following formula:

[0041] The molar ratio of lysine to hyaluronic acid disaccharide units [Lys / HA (mol / mol)] is calculated using the following formula:

[0042] Example 2 Infrared analysis of hyaluronic acid-lysine-copper chelate Infrared analysis was performed on hyaluronic acid-lysine copper chelates (samples 1-3 and 1-9 of Example 1 of this invention) bound by both salt formation and amide bonds using potassium bromide pelleting. The results were compared with the infrared spectra of sodium hyaluronate. (See attached figures.) Figures 1-4 .

[0043] Analysis of the above spectra: A comparison of the IR spectra of hyaluronic acid-lysine copper chelate linked by amide bonds and sodium hyaluronate reveals the following effects: 1) Due to the amide bond between lysine and the carbonyl group of hyaluronic acid, the chemical bonds cause the following effects: 1) The stretching vibration of OH broadens the spectral band, shifting the 3385 cm⁻¹ peak in hyaluronic acid to approximately 3420 cm⁻¹. 2) The stretching vibration of CH causes the 2895 cm⁻¹ peak in hyaluronic acid to shift to approximately 2929 cm⁻¹.

[0044] Comparison of the IR spectra of hyaluronic acid-lysine copper chelate bound by salt formation with sodium hyaluronate shows that, due to the presence of lysine copper salt, 1) its OH stretching vibration and CH stretching vibration are at 3445 cm⁻¹, 3263 cm⁻¹, 3045 cm⁻¹, and 2946 cm⁻¹, respectively. 2) The asymmetric C=O stretching vibration is at 1661 cm⁻¹ and 1585 cm⁻¹, respectively, exhibiting characteristic peaks similar to Lys-Cu-Lys. [References: Liu Feifei, Li Qun, Yu Lan, Cu 2＋ Synthesis and characterization of -Lys chelates. Applied Chemical Industry, 2009, 38(11):1631-1634]. 3) Its fingerprint region at 613 cm⁻¹ still exhibits the characteristic peak of HA.

[0045] Example 3: Inhibitory effect of hyaluronic acid-lysine-copper chelate on 5α-reductase According to the literature method (Pan Jifei et al. Establishment and application of in vitro evaluation system for 5α-reductase inhibitors. Daily Chemical Industry (Chinese and English), 2023, 53(11):1280-1284), the relative inhibition rates of hyaluronic acid lysine copper chelate (HA-lys-Cu) (samples 1-9 of Example 1 of this invention), copper sulfate (CuSO4), and lysine hydrochloride copper chelate (lys-Cu-lys·2HCl) against 5α-reductase (5AR) were determined, and their oil-controlling efficacy was evaluated in vitro. The three compounds were dissolved in water and diluted to a solution containing 0.5 mM copper ions as the test sample. The positive control was a 0.2 mM dutasteride (DTS) solution as a reference. The relative inhibition rates were determined and calculated. The results ( Figure 5The results showed that all three test samples had a significant inhibitory effect on 5α-reductase, and the inhibitory effect of the hyaluronic acid-lysine copper chelate of this invention was better than that of copper sulfate and lysine hydrochloride copper chelate. The in vitro oil-control efficacy evaluation results indicated that, compared with inorganic copper salts—copper sulfate and small molecule organic copper compounds—lysine hydrochloride copper chelate, the oil-control effect of the hyaluronic acid-lysine copper chelate of this invention was improved.

[0046] Example 4: Skin Adhesion of Hyaluronic Acid Lysine Copper Chelate Appropriate amounts of lysine-copper hydrochloride chelate, copper hyaluronic acid (sample 2-1 of Example 1 of this invention), and hyaluronic acid-lysine-copper chelate of this invention (samples 1-3 of Example 1 of this invention) were weighed and dissolved in water to prepare a solution with a copper ion content of 6.87 mg / ml. A 2cm × 2cm test area was selected on the skin, and 0.3 ml of each of the above-mentioned lysine-copper hydrochloride chelate, copper hyaluronic acid, and hyaluronic acid-lysine-copper chelate solutions were applied. After drying, the test area was wiped with a cotton swab every 5 minutes, and the copper ion content wiped away by the cotton swab was measured. Compared with the total amount of copper ions applied to the test area, the cumulative loss of copper ions after multiple wipings (%) was calculated. Results ( Figure 6 The results showed that after 12 wiping cycles, the cumulative loss of copper in the lysine hydrochloride copper chelate was 95.68%, copper hyaluronic acid was 86.68%, and the loss of copper hyaluronic acid lysine copper chelate was 71.50%. This indicates that the skin adhesion of the hyaluronic acid lysine copper chelate of this invention is better than that of the lysine hydrochloride copper chelate and copper hyaluronic acid.

Claims

1. The application of a hyaluronic acid-lysine-copper chelate in the preparation of topical skin products with oil-controlling effects, characterized in that: ① The copper ion content is 5%~7%g / g; ② The molar ratio of lysine to copper ions is 1:0.4–0.6; The molar ratio of hyaluronic acid disaccharide units to lysine is 1:0.2–1.5; The average molecular weight of the hyaluronic acid is 200kDa to 500kDa; The hyaluronic acid-lysine-copper chelate is composed of lysine-copper chelate and hyaluronic acid bonded together by an amide bond, and is represented by general formula (2). （2） Where X, Y, and Z represent the number of hyaluronic acid disaccharide units, X>Y>Z.

2. The application of a hyaluronic acid-lysine-copper chelate in the preparation of cosmetics with oil-controlling effects, characterized in that: ① The copper ion content is 5%~7%g / g; ② The molar ratio of lysine to copper ions is 1:0.4–0.6; The molar ratio of hyaluronic acid disaccharide units to lysine is 1:0.2–1.5; The average molecular weight of the hyaluronic acid is 200kDa to 500kDa; The hyaluronic acid-lysine-copper chelate is composed of lysine-copper chelate and hyaluronic acid bonded together by an amide bond, and is represented by general formula (2). （2） Where X, Y, and Z represent the number of hyaluronic acid disaccharide units, X>Y>Z.

3. The application of a hyaluronic acid-lysine-copper chelate in the preparation of medical products with oil-controlling effects, characterized in that: ① The copper ion content is 5%~7%g / g; ② The molar ratio of lysine to copper ions is 1:0.4–0.6; The molar ratio of hyaluronic acid disaccharide units to lysine is 1:0.2–1.5; The average molecular weight of the hyaluronic acid is 200kDa to 500kDa; The hyaluronic acid-lysine-copper chelate is composed of lysine-copper chelate and hyaluronic acid bonded together by an amide bond, and is represented by general formula (2). （2） Where X, Y, and Z represent the number of hyaluronic acid disaccharide units, X>Y>Z.

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