CN110623889A - Application of lily bulb extract - Google Patents

Application of lily bulb extract Download PDF

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Publication number
CN110623889A
CN110623889A CN201810644100.6A CN201810644100A CN110623889A CN 110623889 A CN110623889 A CN 110623889A CN 201810644100 A CN201810644100 A CN 201810644100A CN 110623889 A CN110623889 A CN 110623889A
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gene
extract
lilium
skin
expression
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Inventor
姜丹
姚期凤
宋肖洁
吴越
蒋耀全
章漳
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Galan (group) Co Ltd
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Galan (group) Co Ltd
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Priority to CN201810644100.6A priority Critical patent/CN110623889A/en
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    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K8/00—Cosmetics or similar toiletry preparations
    • A61K8/18—Cosmetics or similar toiletry preparations characterised by the composition
    • A61K8/96—Cosmetics or similar toiletry preparations characterised by the composition containing materials, or derivatives thereof of undetermined constitution
    • A61K8/97—Cosmetics or similar toiletry preparations characterised by the composition containing materials, or derivatives thereof of undetermined constitution from algae, fungi, lichens or plants; from derivatives thereof
    • A61K8/9783—Angiosperms [Magnoliophyta]
    • A61K8/9794—Liliopsida [monocotyledons]
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61Q—SPECIFIC USE OF COSMETICS OR SIMILAR TOILETRY PREPARATIONS
    • A61Q19/00—Preparations for care of the skin
    • A61Q19/08—Anti-ageing preparations
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K2800/00—Properties of cosmetic compositions or active ingredients thereof or formulation aids used therein and process related aspects
    • A61K2800/80—Process related aspects concerning the preparation of the cosmetic composition or the storage or application thereof
    • A61K2800/84—Products or compounds obtained by lyophilisation, freeze-drying

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  • Health & Medical Sciences (AREA)
  • Life Sciences & Earth Sciences (AREA)
  • Veterinary Medicine (AREA)
  • Public Health (AREA)
  • General Health & Medical Sciences (AREA)
  • Animal Behavior & Ethology (AREA)
  • Microbiology (AREA)
  • Birds (AREA)
  • Epidemiology (AREA)
  • Mycology (AREA)
  • Botany (AREA)
  • Biotechnology (AREA)
  • Engineering & Computer Science (AREA)
  • Gerontology & Geriatric Medicine (AREA)
  • Dermatology (AREA)
  • Cosmetics (AREA)
  • Medicines Containing Plant Substances (AREA)

Abstract

The invention discloses an application of a lilium candidum extract, in particular to an application in preparing an expression up-regulator of a skin aging resistant gene. The anti-skin aging gene can be one or more of eln gene, col3a1 gene, csf2 gene, igf1 gene and nid1 gene. The expression up-regulator of the anti-skin aging gene can enhance and support the stability of the skin tissue structure, increase the thickness of the skin epidermis and delay skin aging, and can be used in cosmetics or skin care products.

Description

Application of lily bulb extract
Technical Field
The invention relates to the field of plant extracts and daily chemicals, in particular to application of a lilium candidum extract.
Background
The skin is covered on the whole human body surface, is in direct contact with the external environment, protects the body from being damaged by various external factors, and is a mirror reflecting the health state of the whole body. The skin is composed of epidermis, dermis, and subcutaneous tissue, and contains skin appendages, abundant nerves, blood vessels, lymphatic vessels, and muscles. The junction area between the dermis and epidermis, also known as the basement membrane zone, is not only a structural model but also has a versatile function, the basement membrane is the supporting cushion of epithelial cells, and serves for structural connection between epithelial cell tissue and connective tissue, as well as other functions. For example, the basement membrane acts as a selective cellular barrier, generally preventing connective tissue cells from contacting epithelial cells on the basement membrane, but allowing lymphocytes, macrophages, and neuronal synapse to cross; in addition, many vital phenomena such as cell morphology, cell polarity, cell metabolism, protein distribution in plasma membranes, cell survival, cell proliferation and differentiation, and cell migration are very closely related to the basement membrane.
Elastin eln (elastin) is an insoluble macromolecular fibrous protein in the extracellular matrix that, together with a structural glycoprotein known as "microfibrils", constitutes the elastic fibers in connective tissue. Elastin stretches several times longer than it is and returns to its original state without the body consuming energy. Thus, it shares the name "human rubber", and elastin is therefore so named. Due to the biological characteristics of elastin, which give elasticity to tissues, certain elasticity-based organ functions such as ligaments, arteries, lungs, skin, etc. are achieved. Morphological studies of human skin biopsies reveal the effect of elastin degradation on skin aging. After elastin degrades, skin loses elasticity, relaxes, wrinkles and other aging phenomena occur. Therefore, increasing skin fibroblasts to synthesize functionally sound elastin, reconstructing elastic fiber structure to resist elastin degradation, and restoring skin elasticity are one of the important ways to delay skin aging.
The col3a1(collagen type III alpha 1chain) gene encodes type III collagen, which encodes the pre-alpha 1chain of type III collagen, a fibrillar collagen, usually associated with type I collagen, and is found in the connective tissues of the extendible body such as the skin, lungs, uterus, intestine and vascular system. The generation of type III collagen helps to strengthen and support the stability of the skin tissue structure and delay skin aging.
Insulin-like growth factor igf1(insulin like growth factor 1) is encoded by this gene as insulin-like growth factor 1, a protein that is functionally and structurally similar to insulin and is a member of a family of proteins involved in regulating growth and development. Insulin-like growth factor 1 has the effects of promoting keratinocyte migration, promoting wound re-epithelialization and influencing the growth and development of skin cells, and the up-regulation of the level of insulin-like growth factor 1 is helpful for delaying aging.
The protein coded by the gene of colony stimulating factor csf2(colony stimulating factor 2) is colony stimulating factor 2, also called granulocyte macrophage colony stimulating factor, is a broad-spectrum cytokine, and has the function of promoting proliferation of early hematopoietic progenitor cells. Mainly acts on myeloid progenitor cells, exerts biological effect by binding with high affinity receptors thereof, enables the cells to rapidly enter the cell cycle and differentiate towards the granulocyte lineage and macrophage lineage to finally become mature cells, can prolong the life of the mature cells, and enhances the functions of the mature cells. Colony stimulating factor 2 is an important cell longevity factor, and is closely related to skin aging.
The Nidogen nidi1(Nidogen1) gene encodes Nidogen, which is an important protein of DEJ, plays a role in connecting type IV collagen and laminin 5 and is essential for the structure and function of DEJ. The structural and functional normality of DEJ determines whether the skin is normally renewed and differentiated. The nestin nidi1(Nidogen1) gene encodes a nestin family member of basement membrane glycoproteins. Proteins interact with several other components of the basement membrane and may play a role in cellular interactions with the extracellular matrix. Thus, the expression of the nidogen nidi1 gene is closely associated with skin aging.
Therefore, the research on how to improve the expression of genes related to key parts and functions of the skin has important significance for improving the stability of the true epidermal structure of the skin and delaying skin aging.
Lilium Candidum (Lilium Candidum) is a perennial herb of Liliales, Liliaceae, Lilium. The bulb is spherical. The stem is upright, the height is more than 1 meter, and the green color has purple stripes. Living under mountain slopes or stream sides. It is distributed in southeast and southwest of China, Henan, Hebei, Shaanxi, Gansu provinces, etc., and also in southern Europe. Perennial herbaceous plants. Bulb-shaped, white, scattered leaves, regularly smaller upper leaves than middle leaves, inverted needle-shaped, 1 to 4 flowers, trumpet-shaped, fragrant, and most of the flower quilt sheet 6 is white in inverted egg shape. The flowering period is 6-8 months, and the capsule is round and has edges and a plurality of seeds. The bulb is expanded, nodeless and white, the stem has purple stripes, the leaves are scattered, the upper leaves are always smaller than the middle leaves, the leaves are inverted needle-shaped, the base part is inclined and narrow, the whole edge is provided with 3 to 5 veins, 1 to 4 flowers are in a trumpet shape, the flower quilt is fragrant, the flower quilt is 6 in an inverted egg shape, most of the flowers are white, the back surface is purple brown, and no spots exist. The top end is bent but not rolled, two sides of the honey gland are provided with small papillae, the male core is bent forwards and grows at the base of the quilt; the filaments are provided with soft hair, the anther is oval, the T-shaped buds grow, the ovary is long and cylindrical, the stigma is 3 cracked, the flowering period is 6-8 months, the capsule is rectangular and has edges, and the majority of seeds are contained.
Disclosure of Invention
The invention aims to solve the technical problem of providing an application of a lilium candidum extract, in particular to an application in preparing an expression up-regulator of an anti-skin-aging gene. The expression up-regulator of the anti-skin aging gene can enhance and support the stability of the skin tissue structure, increase the thickness of the skin epidermis and delay skin aging, and can be used in cosmetics or skin care products.
The invention provides an application of a lilium candidum extract in preparing an expression up-regulator of an anti-skin-aging gene.
The anti-skin aging gene can be one or more of eln gene, col3a1 gene, csf2 gene, igf1 gene and nid1 gene.
The lilium candidum extract can be prepared by extracting lilium candidum with a solvent which is conventional in the field, ethanol and/or water, or water (namely water extract of lilium candidum).
The lilium candidum extract can be an extract of whole plant of lilium candidum, and can also be an extract of any part of lilium candidum, such as an extract of bulbs of lilium candidum.
The preparation method of the lilium candidum extract can be a conventional method in the field, and can also comprise the following steps:
(1) adding water into the powder of the lilium brownii for extraction, and filtering to obtain filtrate;
(2) drying the filtrate.
In the step (1), the powder of the lilium candidum can be powder of lilium candidum bulbs.
In the step (1), the mesh number of the lily bulb powder can be 20-60 meshes, for example 20 meshes.
In the step (1), the amount of the water added may be 15 to 30 times, for example, 20 times, the volume of the powder of the lilium albus.
In step (1), the temperature of the extraction may be 90-100 ℃, for example 96 ℃.
In step (1), the number of times of extraction may be 1 or more, for example, 2 times.
In step (1), the extraction time may be 0.5 to 2 hours, for example, 1 hour.
In step (1), the filtration method may be centrifugation. The centrifugation speed may be 6000-10000rpm, for example 8000 rpm. The centrifugation time may be 8-12 minutes, for example 10 minutes.
In the step (2), the drying may be freeze drying.
In some embodiments of the present invention, the preparation of the lilium candidum extract may comprise the following steps:
(1) adding 20 times of water into the powder of Bulbus Lilii Viriduli, heating in 96 deg.C water bath for 2 times (each for 1 hr), centrifuging, filtering, and mixing filtrates; the mesh number of the powder of the lily bulb is 20 meshes; the speed of the centrifugation is 8000rpm, and the time of the centrifugation is 10 minutes;
(2) and (5) freezing and drying the filtrate.
The concentration of the lilium candidum extract in the expression up-regulator of the skin aging resistant gene can be 8-120 mu g/mL, such as 10-100 mu g/mL.
The concentration of the lilium candidum extract in the expression up-regulator aiming at eln gene can be 8-120 mug/mL, such as 10-100 mug/mL.
The concentration of the lilium candidum extract in the expression up-regulator for one or more of the col3a1 gene, the csf2 gene and the igf1 gene can be 8-12 mug/mL, for example 10 mug/mL.
The concentration of the lilium candidum extract in the expression up-regulator for the nid1 gene can be 80-120 mu g/mL, such as 100 mu g/mL.
Wherein, the expression up-regulator of the anti-skin aging gene can be used in cosmetics or skin care products.
The lilium candidum extract can be used in cosmetics or skin care products.
On the basis of the common knowledge in the field, the above preferred conditions can be combined randomly to obtain the preferred embodiments of the invention.
The reagents and starting materials used in the present invention are commercially available.
The positive progress effects of the invention are as follows: the invention provides an application of a lilium candidum extract, in particular to an application in preparing an expression up-regulator of an anti-skin-aging gene. The expression up-regulator of the anti-skin aging gene can enhance and support the stability of the skin tissue structure, increase the thickness of the skin epidermis and delay skin aging, and can be used in cosmetics or skin care products.
Drawings
FIG. 1 shows the fast blue staining results of 3D skin model section of control group
FIG. 2 shows the fast blue staining results of 10ug/mL Lilium Candidum extract on 3D skin model section
FIG. 3 shows the fast blue staining results of a 3D skin model section of a 100ug/mL Lilium Candidum extract
FIG. 4 shows that 10ug/mL Lilium Candidum extract promotes eln gene expression
FIG. 5 shows that 10ug/mL Lilium Candidum extract promotes the expression of col3a1 gene
FIG. 6 shows that 10ug/mL Lilium Candidum extract promotes expression of csf2 gene
FIG. 7 shows that 10ug/mL Lilium Candidum extract promotes igf1 gene expression
FIG. 8 shows that 100ug/mL Lilium Candidum extract promotes eln gene expression
FIG. 9 shows that 100ug/mL Lilium Candidum extract promotes the expression of nid1 gene
Detailed Description
The invention is further illustrated by the following examples, which are not intended to limit the scope of the invention. The experimental methods without specifying specific conditions in the following examples were selected according to the conventional methods and conditions, or according to the commercial instructions.
Example 1 preparation of Lilium Candidum extract
Taking 10g of lily bulb, crushing to about 20 meshes, adding deionized water with 20 times volume to immerse, heating and extracting for 1h in 96 ℃ water bath, centrifuging (8000rpm, 10min) and filtering to obtain filtrate, repeating the above extraction steps once, combining the filtrates, and freeze-drying the filtrate to obtain about 2.5g of lily bulb extract.
Example 2 preparation of skin model
Extracting human primary fibroblast (NHDF) and human primary keratinocyte (NHEK) from skin tissue (extraction method reference: Wujing, Zhutangyou, skin tissue engineering)
[M]Beijing: civil and military medical press, 2009: 72-103). Reviving the human primary fibroblast, wherein the reviving process comprises the following steps: taking out the freezing tube from the liquid nitrogen, rapidly putting into water bath at 37 ℃ for melting, and centrifuging at 1000rpm/min by a centrifuge for 10 min. Culturing the cells obtained by resuscitation in a carbon dioxide incubator at 37 ℃ for one week by using a DMEM culture medium, then digesting (taking the cells out of the incubator, removing the original culture medium, adding PBS (15 mL) to rinse the cells once, adding 8mL of 0.05% trypsin, incubating for 8min in the carbon dioxide incubator at 37 ℃, taking out, adding 10mL of the DMEM culture medium containing FBS in a super clean bench, taking out the cells in a 50mL centrifuge tube by using a pipette gun, centrifuging for 10min at 1000rpm in a centrifuge, removing supernatant, and standing for use), mixing with collagen for dermal stage culture, culturing for 3-4 days in the carbon dioxide incubator at 37 ℃, and then using a feederHuman primary keratinocytes cultured in layers, digested and inoculated (5X 10)4Individual cells) to the surface of the Skin model in the dermis stage, and culturing for 18 days sequentially through the epidermis submerged culture and gas-liquid phase culture stages to obtain the 3D Skin model for detection (the specific steps for preparing the Skin model are shown in ANNIE F. BLACK, Ph. D. CHARBEL BOUEZ, al. optimization and charateristic of an Engineered Human Skin equivalent. TISSUE ENGINEERING Volume 11, Number 5/6,2005.Mary Ann Liebert, Inc.).
Example 3 Effect test of Lilium Candidum extract
1.1 culture of skin models
The lily alba extract prepared in example 1 is used as a solvent to prepare a lily alba extract solution with a final concentration of 10mg/mL, the extract solution is added according to a test concentration after the skin model is cultured for 3 days, a model without the added extract solution is used as a control, after 18 days, the 3D skin model is sliced, fast blue staining is carried out, the influence of the lily alba extract on the skin epidermis is observed, and meanwhile, RNA in the skin models of a test group and a control group is extracted for detection.
1.2 extraction of RNA
Clipping 3D skin model tissue about 2 x 3mm, and placing into 2mL EP tube; adding 1mL of Trizol, adding 2 steel balls, homogenizing by using a tissue disruptor, and carrying out 50Hz ultrasonic treatment for 5 min; 9000
Centrifuging at 4 deg.C for 1min at rpm, and transferring the supernatant into a new 1.5mL EP tube; adding 200 μ L chloroform, slightly inverting for several times, mixing, and standing at room temperature for 5 min; centrifuging at 9000rpm and 4 deg.C for 15 min; transferring the upper water phase (about 400 μ L) into a new 1.5mL EP tube, adding isopropanol with the same volume, mixing, and standing at room temperature for 10 min; centrifuging at 9000rpm and 4 deg.C for 10 min; discarding the supernatant, washing the precipitate with pre-cooled 70% anhydrous ethanol for 1 time, and centrifuging at 8000rpm and 4 deg.C for 5 min; after the supernatant is discarded, the precipitate is air-dried for 5-10 minutes and then dissolved in 30 mu L of DEPC (diethyl pyrocarbonate) water; the RNA concentration was determined spectrophotometrically.
1.3 Gene expression analysis
RNA purification and quantitative analysis of genes were performed according to the QuantiGene R2.0 Plex Assay kit, and gene expression was analyzed using the luminex platform.
1.4 determination of results
In the gene expression analysis test, if the positive group added with the lilium albo-marginatum extract is compared with a blank control group, the expression level of the gene is obviously increased, and the lilium albo-marginatum extract can be concluded to have the effects of promoting the synthesis of extracellular matrix, stabilizing the structural integrity of a basement membrane, stabilizing a skin structure, inducing the expression of genes related to cell repair and aging resistance, improving the stability of the skin structure and delaying aging.
The effect of the lilium candidum extract is summarized below.
1. Effect of Lilium Candidum extract on skin epidermis
Comparing fig. 2, fig. 3 and fig. 1 (fig. 1, fig. 2 and fig. 3 are all obtained by shooting under a microscope, and the microscope magnification is 10 times), it can be known that after the lilium albugineum extract is added, compared with the reconstructed skin model of an untreated group, the lilium albugineum extracts of 10ug/mL and 100ug/mL can effectively increase the thickness of the epidermis of the reconstructed skin model, the cells on the basal layer are arranged more tightly, and the stratum corneum is more completely differentiated. This indicates that the lilium candidum extract has the potential effects of delaying skin aging, and improving the overall vitality and health condition of the skin.
2. Lilium Candidum extract for resisting influence of skin aging gene expression
As shown in fig. 4 and 5, after adding 10 μ g/mL of lilium albo-marginale extract, the expression of the extracellular matrix-related genes eln, col3a1 was up-regulated by 1.65-fold and 1.35-fold in the skin model, respectively, compared to the control group (NT); as shown in fig. 6 and 7, the anti-aging efficacy-associated genes csf2, igf1 were up-regulated in expression by 1.99-fold and 2.58-fold, respectively, in the skin model, as compared to the control group (NT). Therefore, the lilium albo-marginatum extract under the concentration of 10 mu g/mL can improve the expression of extracellular matrix related genes and anti-aging efficacy related genes, and has the effects of improving the stability of skin structures and delaying aging.
As shown in fig. 8 and 9, when 100 μ g/mL of lilium albo-marginatum extract was added, the expression of the gene eln related to extracellular matrix was up-regulated 2.10 times and the expression of the gene nid1 related to basement membrane stability was up-regulated 1.45 times, compared to the control group (NT). From this, it can be concluded that the lilium albo-marginatum extract at a concentration of 100 can induce the expression of genes related to basement membrane stability and genes related to extracellular matrix, and has the effects of improving the stability of the skin structure and delaying aging.

Claims (10)

1. Application of Bulbus Lilii (Lilium candidum) extract in preparing up-regulator for resisting skin aging gene is provided.
2. The use of claim 1, wherein: the anti-skin aging gene is one or more of eln gene, col3a1 gene, csf2 gene, igf1 gene and nid1 gene.
3. The use of claim 1, wherein: the lilium candidum extract is an aqueous extract of lilium candidum.
4. The use of claim 1, wherein: the lilium candidum extract is an extract of lilium candidum bulbs.
5. The use of claim 1, wherein: the preparation method of the lilium candidum extract comprises the following steps:
(1) adding water into the powder of the lilium brownii for extraction, and filtering to obtain filtrate;
(2) drying the filtrate.
6. The use of claim 5, wherein: the powder of the lilium brownii is the powder of the bulb of the lilium brownii;
and/or the mesh number of the lily powder is 20-60 meshes;
and/or, the amount of water added is 15-30 times, e.g., 20 times, the volume of the Lilium Candidum powder;
and/or the temperature of the extraction is 90-100 ℃, such as 96 ℃;
and/or, the extraction times are more than 1;
and/or, the extraction time is 0.5-2 h;
and/or the filtration method is centrifugation;
and/or, the drying is freeze drying.
7. The use of claim 1, wherein: the preparation method of the lilium candidum extract comprises the following steps:
(1) adding 20 times of water into the powder of Bulbus Lilii Viriduli, heating in 96 deg.C water bath for 2 times (each for 1 hr), centrifuging, filtering, and mixing filtrates; the mesh number of the powder of the lily bulb is 20 meshes; the speed of the centrifugation is 8000rpm, and the time of the centrifugation is 10 minutes;
(2) and (5) freezing and drying the filtrate.
8. The use of claim 7, wherein: the concentration of the lilium candidum extract in the expression up-regulator of the skin aging resistant gene is 8-120 mug/mL, such as 10-100 mug/mL.
9. The use of claim 8, wherein: the concentration of the lilium candidum extract in the expression up-regulator aiming at eln gene is 8-120 mug/mL, such as 10-100 mug/mL;
and/or the concentration of the lilium candidum extract in the expression up-regulator for one or more genes of the col3a1 gene, the csf2 gene and the igf1 gene is 8-12 mug/mL, such as 10 mug/mL;
and/or the concentration of the lilium album extract in the expression up-regulator aiming at the nid1 gene is 80-120 mu g/mL, such as 100 mu g/mL.
10. Use according to any one of claims 1 to 9, wherein: the expression up-regulator of the anti-skin aging gene is used in cosmetics or skin care products.
CN201810644100.6A 2018-06-21 2018-06-21 Application of lily bulb extract Pending CN110623889A (en)

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Citations (4)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
JP2011016760A (en) * 2009-07-09 2011-01-27 Noevir Co Ltd Skin care external preparation, oral agent, moisturizing agent, anti-aging agent, bleaching agent, and anti-oxidizing agent containing bulb of lilium plant and/or callus extract as effective ingredient
CN102727406A (en) * 2010-12-17 2012-10-17 强生消费者公司 Compositions comprising lilium candidum extracts and uses thereof
WO2016055600A1 (en) * 2014-10-09 2016-04-14 Patrinove Lilium bulb extract for use thereof in skin disorders or pathologies
CN107496248A (en) * 2017-09-15 2017-12-22 广州赛莱拉干细胞科技股份有限公司 A kind of net white composition and the skin care item containing said composition

Patent Citations (4)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
JP2011016760A (en) * 2009-07-09 2011-01-27 Noevir Co Ltd Skin care external preparation, oral agent, moisturizing agent, anti-aging agent, bleaching agent, and anti-oxidizing agent containing bulb of lilium plant and/or callus extract as effective ingredient
CN102727406A (en) * 2010-12-17 2012-10-17 强生消费者公司 Compositions comprising lilium candidum extracts and uses thereof
WO2016055600A1 (en) * 2014-10-09 2016-04-14 Patrinove Lilium bulb extract for use thereof in skin disorders or pathologies
CN107496248A (en) * 2017-09-15 2017-12-22 广州赛莱拉干细胞科技股份有限公司 A kind of net white composition and the skin care item containing said composition

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