Patent Description:
Since the concept on organic and natural diets is increasingly prevalent, biotechnology companies and food industries have been intensively investing in the development of natural plant-related products. In order to provide a scientific evidence for the health benefits of plant-related product, the analysis of the active ingredients of plants and evaluation of their efficacy have become the focus of product development.

White roselle (Hibiscus sabdariffa cv. ), also referred to as crystal roselle, white roselle flower, or white jade roselle, is a species in the genus Hibiscus in the family Malvaceae. White roselle is an annual herb or a perennial shrub, growing to about <NUM>-<NUM> in tropical and subtropical regions. It can be used to reduce heat and relieve inflammation, refresh and relieve fatigue, regulate blood lipids, reduce blood pressure, and reduce cholesterol.

<NPL>) disclosed a hibiscus tea, which is an herbal tea made as an infusion from crimson or deep magenta-colored calyces (sepals) of the Roselle (Hibiscus sabdariffa) flower. Vora et al. also disclosed that hibiscus tea has been used for the management of high blood pressure, diabetes, high cholesterol, flu and various skin disorders.

In some embodiments of the present invention, a non-therapeutic use of a white roselle (Hibiscus sabdariffa cv. ) extract for preparing a composition for enhancing skin moisturization is provided, where the white roselle extract is obtained by lysing a cell wall of a white roselle calyx by ice crystals and extracting the lysed white roselle calyx with a solvent.

In conclusion, the white roselle extract of any embodiments of the present invention can prepare a composition for enhancing skin moisturization. In other words, the composition has the function of enhancing skin moisturization.

In some embodiments of the present invention, a white roselle extract obtained from the calyx of white roselle (Hibiscus sabdariffa cv. ) has the capability of enhancing skin moisturization. Therefore, the white roselle extract can be used for preparing a composition with the capability of enhancing skin moisturization. The white roselle extract is obtained by lysing a cell wall of a white roselle calyx by ice crystals prior to the extraction of the lysed white roselle calyx with a solvent.

In some embodiments of the present invention, in the process of extraction, the white roselle calyx is frozen at -<NUM>±<NUM>, so that the cell wall of the white roselle calyx is lysed by ice crystals, and the extraction is carried out on the white roselle calyx with the lysed cell wall at <NUM>±<NUM> with water for <NUM>-<NUM>, so as to obtain a primary extract. For example, the white roselle calyx is soaked in water with a volume <NUM> folds of the volume of the calyx at <NUM>±<NUM> for <NUM>-<NUM>.

In some embodiments of the present invention, the white roselle calyx for extraction may be whole or separated into fragments, granules, or powder by physical pre-processing. The physical pre-processing used may include at least one of the following: coarse crushing, chopping, shearing, mashing, and grinding.

In some embodiments of the present invention, the white roselle calyx is frozen at - <NUM>±<NUM> for more than <NUM>, so that the water contained in the calyx forms ice crystals due to rapid freezing. The formed ice crystals can break the cell wall of the calyx to release more active substances.

In some embodiments of the present invention, in the process of extraction, the primary extract may be further filtered to remove impurities, so as to obtain a filtrate. In some embodiments, during the process of extraction, the filtrate may be further concentrated to obtain a concentrate. In some embodiments, during the process of extraction, the concentrate may be further filtered to obtain a concentrated filtrate.

In some embodiments of the present invention, the concentration is carried out at <NUM>±<NUM>.

In other words, the primary extract, the filtrate, the concentrate, or the concentrated filtrate obtained in the process of extraction may be used as a white roselle extract according to actual needs.

In some embodiments of the present invention, the white roselle extract contributes to the enhancement of skin moisturization by increasing an expression level of epidermal keratinocyte structure maintenance-related genes.

In some embodiments of the present invention, the epidermal keratinocyte structure maintenance-related genes include transglutaminase <NUM> (TGM1) gene and keratin (KRT) gene. The KRT gene may be at least one of the following: KRT1 gene, KRT10 gene, and KRT14 gene.

In some embodiments of the present invention, the white roselle extract contributes to the enhancement of skin moisturization by increasing aquaporins.

In some embodiments of the present invention, the white roselle extract contributes to the enhancement of skin moisturization by increasing an expression level of aquaporin genes.

In some embodiments of the present invention, the aquaporin gene may be aquaporin <NUM> (AQP3) gene.

In some embodiments of the present invention, the white roselle extract contributes to the enhancement of skin moisturization by increasing ceramides.

In some embodiments of the present invention, the white roselle extract contributes to the enhancement of skin moisturization by increasing ceramide generation-related genes.

In some embodiments of the present invention, the ceramide generation-related genes include glucosylceramidase (GBA) gene and sphingomyelin phosphodiesterase <NUM> (SMPD1) gene.

In some embodiments of the present invention, the white roselle extract contributes to the enhancement of skin moisturization by increasing hyaluronic acid secretion.

In some embodiments of the present invention, the white roselle extract contributes to the enhancement of skin moisturization by increasing an expression level of hyaluronic acid synthesis-related genes.

In some embodiments of the present invention, the hyaluronic acid synthesis-related gene may be hyaluronan synthase (HAS) gene. The HAS gene includes HAS2 gene and HAS3 gene.

In some embodiments of the present invention, an effective dose of the white roselle extract is <NUM>/day.

In some embodiments of the present invention, the white roselle (Hibiscus sabdariffa) extract can be used for preparing a composition for enhancing skin moisturization, and the composition prepared may be a food composition.

In some embodiments of the present invention, the composition is a food composition containing a specific content of white roselle extract. The food composition may be in the form of powder, granule, solution, colloid, or paste.

In some embodiments of the present invention, the food composition containing the white roselle extract may be a food product or a food additive.

In some embodiments of the present invention, the food product containing the white roselle extract may be beverages, fermented foods, bakery products, health foods, dietary supplements, or the like. In some embodiments, the food product containing the white roselle extract may further include an adjuvant. For example, the adjuvant may be maltodextrin, malic acid, sucralose, citric acid, fruit flavor, honey flavor steviol glycoside, or a combination thereof. The type and quantity of selected carriers are within the expertise and routine of those skilled in the art.

In some embodiments of the present invention, the food additive containing the white roselle extract may be a condiment, a sweetener, a flavor, a pH adjuster, an emulsifier, a colorant, a stabilizer, or the like.

Unless otherwise specified, the experimental steps in the following examples are carried out at room temperature (<NUM>±<NUM>) and atmospheric pressure (<NUM> atm).

HPEK-<NUM> cells were seeded in a <NUM>-well culture plate containing <NUM> of culture medium per well in a density of <NUM>×<NUM><NUM> cells per well, and cultured at <NUM> for <NUM>. After the culture, the HPEK-<NUM> cells were divided into the following three groups: a blank group, an experimental group A, and an experimental group B. The culture medium of the blank group contained no extract; the culture medium of the experimental group A contained <NUM>% (v/v) white roselle extract prepared in Example <NUM>; and the culture medium of the experimental group B contained <NUM>% (v/v) roselle extract prepared in Example <NUM>. Each group was triplicated and then cultured at <NUM> for <NUM>. After the culture, the culture media of the blank group, experimental group A, and experimental group B were removed, and the cells were rinsed with PBS. After the rinse, the cell membranes of the HPEK-<NUM> cells in each group were lysed with a cell lysis buffer from an RNA extraction reagent kit to form a cell solution. In each group, RNA was extracted from the cell solution by using the RNA extraction reagent kit. In each group, <NUM> ng of the extracted RNA was used as a template, and reverse-transcribed with SuperScript® III reverse transcriptase into corresponding cDNA. The quantitative real-time reverse transcription polymerase chain reaction was carried out on the cDNA with the primers in Table <NUM> by using the ABI StepOnePlus™ Real-Time PCR system and the KAPA SYBR FAST qPCR Master Mix (2X) Kit to observe the expression level of various target genes of the HPEK-<NUM> cells in the blank group, experimental group A, and experimental group B, and to obtain a melting curve thereof. The instrument setting conditions for the quantitative real-time reverse transcription polymerase chain reaction were <NUM> for <NUM>, <NUM> for <NUM>, <NUM> for <NUM> with <NUM> repetitive cycles. The relative expression level of the target gene was determined by the <NUM>-ΔΔCt method. The relative expression level was defined as a fold change of the RNA expression level of a target gene in the experimental group relative to the same gene in the blank group. The <NUM>-ΔΔCt method used the cycle threshold (Ct) of the TBP gene as the Ct of the reference gene of the internal control, and calculated the fold change according to the following formula: <MAT> <MAT> <MAT>.

The statistically significant difference of the determination results between the blank group and the experimental group was analyzed by student's t-test. (In the figures, "*" represents a p value less than <NUM> in comparison with the blank group, "**" represents a p value less than <NUM> in comparison with the blank group, and "***" represents a p value less than <NUM> in comparison with the blank group. More "*" represents more significant statistical differences.

Refer to <FIG>. An expression level of genes of the HPEK-<NUM> cells untreated with the white roselle extract or the roselle extract in the blank group, that was, under normal physiological metabolism, was regarded as <NUM>. Compared with the blank group, the relative expression level of TGM1 gene in the experimental group A (with the white roselle extract) was <NUM>, and the relative expression level of TGM1 gene in the experimental group B (with the roselle extract) was <NUM>; the relative expression level of KRT1 gene in the experimental group A (with the white roselle extract) was <NUM>, and the relative expression level of KRT1 gene in the experimental group B (with the roselle extract) was <NUM>; the relative expression level of KRT10 gene in the experimental group A (with the white roselle extract) was <NUM>, and the relative expression level of KRT10 gene in the experimental group B (with the roselle extract) was <NUM>; and the relative expression level of KRT14 gene in the experimental group A (with the white roselle extract) was <NUM>, and the relative expression level of KRT14 gene in the experimental group B (with the roselle extract) was <NUM>.

It can be learned that the white roselle extract, different from the roselle extract, can significantly increase the expression level of the TGM1 gene and KRT1 gene, while the roselle extract reduces the expression level of the TGM1 gene and KRT1 gene. In addition, the white roselle extract can significantly increase the expression level of the KRT10 gene and KRT14 gene, better than the roselle extract.

HPEK-<NUM> cells were seeded in a <NUM>-well culture plate containing <NUM> of culture medium per well in a density of <NUM>×<NUM><NUM> cells per well, and cultured at <NUM> for <NUM>. After the culture, the HPEK-<NUM> cells were divided into the following three groups: a blank group, an experimental group A, and an experimental group B. The culture medium of the blank group contained no extract; the culture medium of the experimental group A contained <NUM>% (v/v) white roselle extract prepared in Example <NUM>; and the culture medium of the experimental group B contained <NUM>% (v/v) roselle extract prepared in Example <NUM>. Each group was triplicated and then cultured at <NUM> for <NUM>. After the culture, the culture media of the blank group, experimental group A, and experimental group B were removed, and the cells were rinsed with PBS. After the rinse, the cell membranes of the HPEK-<NUM> cells in each group were lysed with a cell lysis buffer from an RNA extraction reagent kit to form a cell solution. In each group, RNA was extracted from the cell solution by using the RNA extraction reagent kit. In each group, <NUM> ng of the extracted RNA was used as a template, and reverse-transcribed with SuperScript® III reverse transcriptase into corresponding cDNA. The quantitative real-time reverse transcription polymerase chain reaction was carried out on the cDNA with the primers in Table <NUM> by using the ABI StepOnePlusTM Real-Time PCR system and the KAPA SYBR FAST qPCR Master Mix (2X) Kit to observe the expression level of various target genes of the HPEK-<NUM> cells in the blank group, experimental group A, and experimental group B, and to obtain a melting curve thereof. The instrument setting conditions for the quantitative real-time reverse transcription polymerase chain reaction were <NUM> for <NUM>, <NUM> for <NUM>, <NUM> for <NUM> with <NUM> repetitive cycles. The relative expression level of the target gene was determined by the <NUM>-ΔΔCt method. The relative expression level is defined as a fold change of the RNA expression level of a target gene in the experimental group relative to the same gene in the blank group. The <NUM>-ΔΔCt method used the cycle threshold (Ct) of the TBP gene as the Ct of the reference gene of the internal control, and calculated the fold change according to the following formula: <MAT> <MAT> <MAT>.

Refer to <FIG>. An expression level of genes of the HPEK-<NUM> cells untreated with the white roselle extract or the roselle extract in the blank group, that was, under normal physiological metabolism, was regarded as <NUM>. Compared with the blank group, the relative expression level of AQP3 gene in the experimental group A (with the white roselle extract) was <NUM>, and the relative expression level of AQP3 gene in the experimental group B (with the roselle extract) was <NUM>.

It can be learned that the white roselle extract can significantly increase the expression level of the AQP3 gene, better than the roselle extract.

HPEK-<NUM> cells were seeded in a <NUM>-well culture plate containing <NUM> of culture medium per well in a density of <NUM>×<NUM><NUM> cells per well, and cultured at <NUM> for <NUM>. After the culture, the HPEK-<NUM> cells were divided into the following three groups: a blank group, an experimental group A, and an experimental group B. The culture medium of the blank group contained no extract; the culture medium of the experimental group A contained <NUM>% (v/v) white roselle extract prepared in Example <NUM>; and the culture medium of the experimental group B contained <NUM>% (v/v) roselle extract prepared in Example <NUM>. Each group was triplicated and then cultured at <NUM> for <NUM>. After the culture, the culture media of the blank group, experimental group A, and experimental group B were removed, and the cells were rinsed with PBS. After the rinse, the cell membranes of the HPEK-<NUM> cells in each group were lysed with a cell lysis buffer from an RNA extraction reagent kit to form a cell solution. In each group, RNA was extracted from the cell solution by using the RNA extraction reagent kit. In each group, <NUM> ng of the extracted RNA was used as a template, and reverse-transcribed with SuperScript® III reverse transcriptase into corresponding cDNA. The quantitative real-time reverse transcription polymerase chain reaction was carried out on the cDNA with the primers in Table <NUM> by using the ABI StepOnePlus™ Real-Time PCR system and the KAPA SYBR FAST qPCR Master Mix (2X) Kit to observe the expression level of various target genes of the HPEK-<NUM> cells in the blank group, experimental group A, and experimental group B, and to obtain a melting curve thereof. The instrument setting conditions for the quantitative real-time reverse transcription polymerase chain reaction were <NUM> for <NUM>, <NUM> for <NUM>, <NUM> for <NUM> with <NUM> repetitive cycles. The relative expression level of the target gene was determined by the <NUM>-ΔΔCt method. The relative expression level is defined as a fold change of the RNA expression level of a target gene in the experimental group relative to the same gene in the blank group. The <NUM>-ΔΔCt method used the cycle threshold (Ct) of the TBP gene as the Ct of the reference gene of the internal control, and calculated the fold change according to the following formula: <MAT> <MAT> <MAT>.

Refer to <FIG>. An expression level of genes of the HPEK-<NUM> cells untreated with the white roselle extract or the roselle extract in the blank group, that was, under normal physiological metabolism, was regarded as <NUM>. Compared with the blank group, the relative expression level of SMPD1 gene in the experimental group A (with the white roselle extract) was <NUM>, and the relative expression level of SMPD1 gene in the experimental group B (with the roselle extract) was <NUM>; and the relative expression level of GBA gene in the experimental group A (with the white roselle extract) was <NUM>, and the relative expression level of GBA gene in the experimental group B (with the roselle extract) was <NUM>.

It can be learned that the white roselle extract can significantly increase the expression level of the SMPD1 gene and GBA gene, better than the roselle extract.

Refer to <FIG>. An expression level of genes of the HPEK-<NUM> cells untreated with the white roselle extract or the roselle extract in the blank group, that was, under normal physiological metabolism, was regarded as <NUM>. Compared with the blank group, the relative expression level of HAS2 gene in the experimental group A (with the white roselle extract) was <NUM>, and the relative expression level of HAS2 gene in the experimental group B (with the roselle extract) was <NUM>; and the relative expression level of HAS3 gene in the experimental group A (with the white roselle extract) was <NUM>, and the relative expression level of HAS3 gene in the experimental group B (with the roselle extract) was <NUM>.

It can be learned that the white roselle extract, different from the roselle extract, can significantly increase the expression level of the HAS2 gene and HAS3 gene, while the roselle extract reduces the expression level of the HAS2 gene and HAS3 gene.

Refer to <FIG>. An amount of hyaluronic acid produced by the HPEK-<NUM> cells untreated with the white roselle extract or the roselle extract in the blank group, that was, under normal physiological metabolism, was regarded as <NUM>%. Relative to the blank group, an amount of hyaluronic acid produced by the HPEK-<NUM> cells treated with the white roselle extract in the experimental group A was <NUM>%. Relative to the blank group, an amount of hyaluronic acid produced by the HPEK-<NUM> cells treated with the roselle extract in the experimental group B was <NUM>%. In other words, compared with the blank group, the relative hyaluronic acid secretion rates of both the experimental group A and the experimental group B increased. Compared with the blank group, the relative hyaluronic acid secretion of the experimental group A (with the white roselle extract) was increased by <NUM>%. Compared with the blank group, the relative hyaluronic acid secretion of the experimental group B (with the roselle extract) was increased by <NUM>%. It can be learned that both the white roselle extract and the roselle extract can significantly promote the production of hyaluronic acid in HPEK-<NUM> cells, and the capability of white roselle extract to increase hyaluronic acid secretion is better than that of the roselle extract. Based on this, the white roselle extract has the effect of promoting hyaluronic acid secretion, which is beneficial to retain skin collagen, increase skin moisture content, and provide skin elasticity and flexibility.

Hyaluronic acid can prevent natural aging of the skin, and protect the skin from the damage caused by the sun's ultraviolet rays, tobacco smoke, and air pollutants. Hyaluronic acid can also help increase skin moisture, so that the skin structure is firm and plump, so as to reduce skin fine texture and wrinkles. In addition, hyaluronic acid also plays a key role in wound healing. When skin cells need to be repaired or are damaged, the concentration of hyaluronic acid will also increase, and its use on skin wounds has been proven to reduce the size of the wound and relieve pain. It can also help reduce the risk of wound cell infection.

Moreover, hyaluronic acid is also helpful for osteoarthritis. It is shown from literatures that consuming <NUM>-<NUM> of hyaluronic acid every day for at least two months can significantly relieve the knee joint pain in patients with osteoarthritis. Hyaluronic acid can also help relieve gastric acid reflux symptoms. Hyaluronic acid has excellent moisturizing properties, so that it is also commonly used to treat dry eye syndrome, slow down osteoporosis, relieve bladder pain syndrome, and the like.

Nine healthy adult subjects aged <NUM>-<NUM> were asked to drink a <NUM> white roselle extract drink (containing <NUM> of the white roselle extract prepared in Example <NUM>) every day for four weeks (equal to <NUM> days).

Before drinking (the face was clean, week <NUM>), after drinking for <NUM> days (the face was clean, week <NUM>), and after drinking for <NUM> days (the face was clean, week <NUM>), skin detection and skin condition questionnaire were carried out. The skin detection is to record values of the facial skin by corresponding devices and measurement methods, and to take photos before and after drinking. (When the detection was carried out before and after drinking, the temperature and humidity of the detection region where the subjects were located were consistent to reduce the influence of external temperature and humidity on the skin).

The facial skin of the same subject was detected before drinking the white roselle extract drink, after drinking for <NUM> days, and after drinking for <NUM> days by using a skin moisture content detection probe Corneometer® CM825 (C+K Multi Probe Adapter System, Germany) purchased from Courage+Khazaka electronic, Germany. The detection probe is based on the principle of capacitance measurement. When the moisture content changes, the capacitance value of the skin also changes, so that the moisture content of the skin surface can be analyzed by measuring the capacitance value of the skin.

The facial skin of the same subject was detected before drinking the white roselle extract drink, after drinking for <NUM> days, and after drinking for <NUM> days by using a TEWL detection probe Tewameter® TM <NUM> (C+K Multi Probe Adapter System, Germany) purchased from Courage+Khazaka electronic, Germany. The detection probe uses a cylindrical cavity with open ends to form a relatively stable test environment on the skin surface, and measures the water vapor pressure gradient at two different points to calculate the amount of water evaporated through the epidermis, so as to measure the water loss on the skin surface.

The facial skin of the same subject was detected before drinking the white roselle extract drink, after drinking for <NUM> days, and after drinking for <NUM> days by using a skin physiological detector Soft Plus purchased from Callegari <NUM>, Italy. The test principle of the detector is that, based on the principle of suction and stretching, a negative pressure is generated on the surface of the skin to suck the skin into a test probe, the depth of the skin sucked into the probe is detected through the optical test system, and the skin elasticity is calculated by software analysis.

The facial skin of the same subject was detected before drinking the white roselle extract drink, after drinking for <NUM> days, and after drinking for <NUM> days by using a VISIA high class digital skin quality detector (VISIA Complexion Analysis System) purchased from Canfield scientific, US. The test principle of the detector is that the facial skin is photographed through a high-resolution camera lens, and the change of the skin shadow is detected by standard white light irradiation to detect the texture position and obtain a value that can represent the smoothness of the skin.

The facial skin of the same subject was detected before drinking the white roselle extract drink, after drinking for <NUM> days, and after drinking for <NUM> days by using a high-frequency ultrasound detection probe (High Freq. Ultrasound Module) (DermaLab® USB Skin Analyzer, Denmark) purchased from Cortex Technology, Denmark. The detection probe transmitted acoustic pulses into the skin and converted the reflected signals of different intensities into different color markers. The lighter or brighter color indicates more collagen in the skin.

The skin condition questionnaire was carried out on the same subject before drinking the white roselle extract drink, after drinking for <NUM> days, and after drinking for <NUM> days. The skin condition questionnaire is self-assessment for dry and itchy skin, skin sagging, and lack of skin elasticity.

It is to be noted that the statistically significant difference of the determination between week <NUM> and week <NUM> and between week <NUM> and week <NUM> was analyzed by student's t-test. In the figures, "*" represents a p value less than <NUM> in comparison with week <NUM>, "**" represents a p value less than <NUM> in comparison with week <NUM>, and "***" represents a p value less than <NUM> in comparison with week <NUM>. More "*" represents more significant statistical differences.

Claim 1:
A non-therapeutic use of a white roselle (Hibiscus sabdariffa cv.) extract for preparing a composition for skin moisturization, characterized in that the white roselle extract is obtained by lysing a cell wall of a white roselle calyx by ice crystals at -<NUM>±<NUM> and extracting the lysed white roselle calyx with a solvent at <NUM>±<NUM>, wherein the composition for skin moisturization is ingested by a human subject.