JPH02222676A - Crossbred cell, production thereof and cosmetic - Google Patents

Abstract

NEW MATERIAL:A crossbred cell of sponge cucumber and Gynostemma pentaphyllum Makino obtained by cell fusion of protoplast of sponge cucumber and protoplast of Gynostemma pentaphyllum Makino. USE:Used for production of cosmetics having skin conditioning effect. PREPARATION:For instance, seed of sterilized sponge cucumber is germinated on agar-agar and grown on the medium, then cotyledon is cut to strip-like, thus dipped in enzyme solution and incubated to isolate protoplast of cotyledon of sponge cucumber. On the other hand, section of stem of cultured Gynostemma pentaphyllum Makino is sterilized and cultured on medium to induce callus, then dipped in enzyme solution, thus incubated to obtain protoplast of cultured cell of Gynostemma pentaphyllum Makino. Next, said two protoplasts are mixed and subjected to cell fusion with adding of polyethylene glycol, then cultured, thus dipped in enzyme solution and incubated to isolate protoplast of fused cell, then said cell is cultured to afford the aimed crossbred cell.

Description

DETAILED DESCRIPTION OF THE INVENTION (Industrial Application Field) The present invention relates to hybrid cells of loofah and Jiaogulan, a method for producing the same, and a cosmetic containing the extract as an active ingredient.

(Prior technology) In plants such as Brassicaceae, Solanaceae, Apiaceae, Poaceae, and Rutaceae, it is possible to create hybrid cells and hybrid plants that cannot be obtained by conventional hybridization methods by cell fusion between species or in Wakkanai. Many cases have been reported.

On the other hand, it has been reported that extracts from tissue cultures of Cucurbitaceae plants are useful as cosmetic base materials (Japanese Unexamined Patent Publication No. 62-2
73).

(Problems to be Solved by the Invention) In the Cucurbitaceae family, there are no reports of hybrid cells being obtained by cell fusion.

On the other hand, when cultured products of Cucurbitaceae plants are used, for example, in cosmetics, products with higher effectiveness are desired.

Therefore, this time, we fused protoplasts of loofah, a member of the Cucurbitaceae family, with protoplasts of Jiaogulan.
They obtained hybrid cells that proliferated more vigorously than cultured loofah or Jiaogulan cells, and discovered that the extract showed higher cell proliferation activity against human epithelial cells. The present invention was completed by discovering that a cosmetic containing the following has an excellent skin conditioning effect.

(Means for Solving the Problems) That is, the present invention provides hybrid cells obtained by cell fusion of loofah protoplasts and Jiaogulan protoplasts, a method for producing the hybrid cells, and a cosmetic containing an extract of the hybrid cells as an active ingredient. be.

In the present invention, the loofah is a loofah (Lufa) of the Cucurbitaceae family, the genus Loofah.
ffi cylindrica Roes,)
Luffa acutangula
Raxb, ) and also.

Gynostemma is a Gynostemma genus of the Cucurbitaceae family.
Makino).

The cotyledons or cultured cells of loofah are preferably used as materials for isolating protoplasts of loofah.

The preferred material for isolation of Jiaogulan protoplasts is cultured cells thereof.

Loofah cotyledons can be obtained, for example, from seedlings that are grown by aseptically germinating loofah seeds that have been sterilized by a conventional method. In addition, cultured loofah cells are, for example,
0.1-1.0ppI11 dinitrophenoxyacetic acid (2
, 4-D), 0.1-1. It can be obtained by placing sections of loofah cotyledons, hypocotyls, etc. on a Murashige & Skoog medium (MS medium) containing Oppm kinetin and culturing them.

On the other hand, for cultured cells of Jiaogulan, for example, 0.1 to 1.0 pp of Jiaogulan stem sections sterilized by a conventional method
m-bichloram, 0.1-1. It can be obtained by placing it on an MS medium containing Oppm kinetin and culturing it.

The cultured cells used for protoplast isolation are preferably those that have been subcultured for several months or years and have stable characteristics.

Protoplasts are produced by a conventional method, that is, by immersing or suspending the above materials in an enzyme solution containing cellulase, pectinase, an osmotic pressure regulator, for example, mannitol, and incubating at 25°C for about 1 to 20 hours. can do.

Cell fusion can be achieved by mixing loofah and Jiaogulan protoplasts and treating them with chemicals, electricity, etc.

The drug is polyethylene glycol (PEG),
Examples include PEG, dimethyl sulfoxide (DMSO), dextran, polyvinyl alcohol, etc.
Fusion can be achieved with the highest fusion rate using EG and DMSO. In that case, the average molecular weight of PEG is about 15
00-9000, preferably about 6000-9
000 is good. In addition, the appropriate concentration of PEG varies depending on the molecular weight, but is preferably about 5 to 30% (Vl/V), and the concentration of DMSO is 5 to 15% (Vl/V).
V/V) level is good.

On the other hand, a high pH range of 9 to 11 during treatment is excellent for fusion, and a calcium ion concentration of 50 to 2
Approximately 00mM is preferable. That is, conditions of high pH and high C& are preferable.

In the case of electrofusion, a commercially available electrofusion device can be used. Protoplasts, for example, O-1
, suspended in a mannitol solution containing calcium ions of about OsM, and the high frequency (AC) is 0.5 to 1.5 M1lz.
to about 50 to 200 V/cm, and then to current (DC)
The pulses were 0.5-3.
It can be set to about OkV/Cm. Cell fusion can be achieved by applying radiofrequency until the protoplasts are oriented and then applying DC pulses several times while monitoring the fusion state.

The operation of cell fusion using drugs or electricity is described in works on cell fusion in plants, for example, published by Atsushi Ii et al. [Biochemistry Experimental Methods 16. You can refer to "Introduction to Plant Cell Breeding, 1983, Gakkai Publishing Center Publishing" and "Genetic Engineering of Protoplasts," written by Toshiyuki Nagata, 1986, Kodansha.

The cell fusion-treated loofah and Jiaogulan protoplast suspension can be cultured in a medium used for protoplast culture. For example, use Murashige &Skoog's medium, Gamborg et al.'s medium, or a modified medium thereof, which contains plant hormones (auxin, cytokinin), osmotic pressure regulator mannitol, etc., at 25°C in the light (approximately 500-5000111X) or Can be cultured in the dark.

In order to select cells that are a fusion of loofah and Jiaogulan (hybrid cells), it is necessary to combine methods such as inactivation of the metabolic system by treatment with iodine compounds and selection by the medium used for culture (hormone requirement, etc.). . For example, loofah protoplasts are treated in advance with iodoacetamide or monoiodoacetic acid to prevent them from dividing, and then fused with Jiaogulan protoplasts, and cultured in a medium containing hormones that allow loofah protoplasts to divide but prevent Jiaogulan protoplasts from dividing. For example, by fusing loofah and Jiaogulan protoplasts, a hybrid cell colony with complementary metabolic systems and hormone requirements can be efficiently obtained.

For the treatment with an iodine compound, protoplasts may be suspended in, for example, a 1-50 mM iodoacetamide or monoiodoacetic acid solution and treated at around 4° C. for about 5-30 minutes.

In addition, without conducting the above selection, the callus finally obtained by cell fusion may be classified as a hybrid callus (! ) can also be selected.

The obtained colony of hybrid cells is subcultured while lowering the concentration of the osmotic pressure regulator in the medium, allowing it to grow and obtain a hybrid callus.

The hybrid callus (hybrid cells) thus obtained may be grown on an agar medium or suspended in a liquid medium and shaken.

The extract of hybrid cells of loofah and Jiaogulan used in the present invention is obtained by extracting the hybrid cells obtained as described above with a solvent.

For example, water, alcohols (methanol, ethanol,
propylene glycol, etc.). Hybrid cells are extracted with one or a mixed solvent of two or more of these solvents. The extraction may be performed by heating or at room temperature, and the extract may contain the solvent or may be a solid after the solvent is distilled off.

The cosmetics of the present invention are Ml of loofah and Jiaogulan! I[M
In addition to the extract of I. algae, raw materials used in cosmetics can be added within a range that does not impair its effectiveness. Cosmetics of the present invention manufactured from these raw materials include basic cosmetics such as lotions, milky lotions, and creams, makeup cosmetics such as foundations and lipsticks, hair cosmetics such as hair liquids, hair tonic, and hair creams. Examples include cleaning products such as shampoos and facial cleansers, and bath additives.

The amount of the hybrid cell extract of loofah and Jiaogulan used in the cosmetic of the present invention, based on the total amount of the cosmetic, is about 0.0001-10% by weight as a solid from which the solvent has been distilled off, preferably 0.0001-10% by weight. 0.005 to 1.0% by weight.

If the blending amount is less than 0.0001fffj1%, sufficient effects cannot be obtained. Moreover, if it exceeds 10% by weight, it is uneconomical.

Next, the present invention will be explained in more detail by showing examples. Note that the present invention is not limited to this.

Example - Generation of 1M seed cells (PEG-DMSO method) 2%
Luffa Cyli-nd sterilized in sodium hypochlorite solution for 20 minutes and subsequently washed with sterile water
rica Roes, ) on agar and further germinated with hormone-free Murashige & Skoog (MS) seeds.
The sterile plants of loofah were grown for 5 days on the culture medium of Asahi.
The cotyledons of this sterile plant were cut into strips and mixed with 0.3% Meishichi Rause P-1 (manufactured by Meiji Seika Co., Ltd.), 0.05% Macerozyme R-10 (manufactured by Yakult Pharmaceutical Co., Ltd.), l
Luffa cotyledon protoplasts were isolated by immersing them in an enzyme solution (pI-15,7) containing MS inorganic salt (1/2M5) and 0.4M mannitol and incubating at 25°C for 20 hours. In order to inactivate the metabolic system of loofah cotyledon protoplasts and completely suppress their division, 1
01! The protoplasts were suspended in a 0.4M mannitol solution containing M iodoacetamide and incubated for 15 minutes at 4°C.

On the other hand, the stem sections of Jiaogulan cultivated products were placed in a 1% sodium hypochlorite solution for 20 minutes [! The material was sterilized, washed with sterile water, and used as a material for inducing callus in Jiaogulan.
Callus was induced by placing on MSm soil containing inetin and 0.2% casamino acids. This Jiaogulan callus was placed in a liquid medium with the same composition. Suspension culture cells were prepared using rotary shaking culture, and the suspension culture cells were subcultured for 2 years.9 The suspension culture cells in the logarithmic growth phase were mixed with 1% driselase (Kyowa Hakko Kogyo Co., Ltd.) company), 1% Cellulase Onozuka” R-10 (manufactured by Yakult Pharmaceutical Co., Ltd.), 0.5% Macerozyme R-10
(manufactured by Yakult Pharmaceutical Co., Ltd.) and suspended in an enzyme solution (pH 5.5) containing 0.4M mannitol at 25°C.
The protoplasts of the cultured Jiaogulan cells were isolated by incubation for 3 hours.The loofah and Jiaogulan protoplasts thus obtained were purified respectively.
5 solution (125mMcacla, 155mM Na
Cl, 5mM KCl.

2XIO' to 51allI glucose, 9115.6)
The Asahi fusion method is based on the method of Menczel et al. (Plant Ce/1).
1l Rep.

3.196-198 (1984)), PEC? J solution (10% polyethylene glycol 6000 (manufactured by Wako Pure Chemical Industries, Ltd., average molecular weight 75)
00), 10% dimethyl sulfoxide (DMSQ,
O,1M glycine (manufactured by Katayama Chemical Industry Co., Ltd.)
A protoplast solution of luffa and Jiaogulan mixed at a ratio of 1 to 2 liters was added dropwise onto Na011 buffer (pH 10) to start a cell fusion reaction.
Pond prepared by the method of Czel et al. After reacting for 0 minutes, 5
0 carbonate M 2-(N-morpholino)ethanesulfonic acid (
After adding W5 solution (pH 5.5) containing MES) and allowing it to stand at room temperature for 2 hours, the protoplasts were collected by centrifugation.
After washing once with medium, the cells were cultured. 1.0pp for the medium
- Using a 172M5 liquid medium (with an inorganic salt concentration of !/2) containing 0.5 ppm of α-naphthalene acetic acid (NA^), 6-benzylaminopurine (BAP), 1% shakerose, and 0.3 M mannitol. In this medium, Jiaogulan cultured cell protoplasts could not divide. The fusion rate (percentage of fused loofah and Jiaogulan cells to remaining protoplasts) was 10-15%.

In order to obtain as many colonies as possible, loofah cotyledon protoplasts that had not been treated with iodoacetamide were cultured by separating them with filter paper, and the fused cells were nurse cultured, and several dozen colonies of approximately 0.5 mm in size were obtained in the lateral zone within about one month after the start of Asahi culture. Ta.

No colonies were formed when iodoacetamide-treated loofah cotyledon protoplasts and Jiaogulan cultured cell protoplasts were simply mixed and cultured without cell fusion. Furthermore, no colonies were formed when each protoplast was individually fused and cultured.

The obtained colonies were subcultured while decreasing the mannitol concentration in the above medium, and six calli were finally obtained.Protoplasts were obtained from these calli in the same manner as when isolating protoplasts from cultured Jiaogulan cells. was isolated.

And each of the obtained protoplasts! ×104
The concentration of the medium is 1. Op
l/2M S with p+w NM, 0.5ppm BAP, 1% Shakrose and 0.3M Mannitol
A gellan gum (0.15%, manufactured by Sanei Chemical Industry Co., Ltd.) medium was used.

Colonies of about 0.5 mm (derived from a single 700ml) were obtained one month after the start of culture, and then subcultured while lowering the amount of mannitol a in the medium to obtain callus. These calluses (F
1, F2, F3, F4, F5, F6) were used for the following analysis, testing, and culture.

Four of these calluses (F3.F4, F5, F
6) showed hybridity in isozyme analysis and hormone requirement tests.

That is, as shown in Figures 1 and 2, in the isozyme analysis of glucose-6-phosphate dehydrogenase, these calli (F3, F4, F5, F6) were found to be the band of loofah cotyledon protoplast-derived callus (B). Furthermore, these calli also showed hybridity in esterase isozyme analysis.As shown in Table 1, calli derived from luffa cotyledon protoplasts had N. ^^
These calli (F3, F4, F5, and F6) were able to grow in both media, whereas cultured cells of Gynostemma and Jiaogulan could only grow in picloram media. .

Table-1 Hormone requirement test medium 1: 1. oppm NM, 0.5ppm BA
MS medium containing P and 0.2% casamino acids Medium 2: 0.5 ppm vichloram, o, tpp ■ MS medium containing kinetin and 0.2% casamino acids +: Proliferated, -2 hardly proliferated. These hybrid calluses are 1. Oppm NAA, 0.5ppm
B.A.P.

It grew very well in MS medium containing 0.2% casamino acids.9 In cell suspension culture using the same liquid medium, 25'
C. Rotary shaking culture (90 r, p, m,) for 10 days under 24-hour illumination (20001 ux) resulted in very fine suspension cultured cells, with a dry rfL amount of 4 to 5 g of cells per liter of culture. These hybrid cells also showed similar growth in a medium in which NAA was replaced with indole acetic acid (1^^). There is no change in growth rate, hormone requirement, morphology, color, or band pattern in isozyme analysis (
, and has very stable genetic properties when cells are fused using the normal PEG method without using DMSO.

Alternatively, this PEG-
Using the DMSO method, cells can be fused with a fusion rate of about 10 to 15%, and hybrid cells of loofah and Jiaogulan can be obtained efficiently and reproducibly.Asahi Example-2 Production of hybrid cells (electrofusion) Method) Luffa cotyledon protoplasts and Jiaogulan cultured cell protoplasts were isolated in the same manner as in Example 1. After purifying these protoplasts, they were each suspended in a 0.3M mannitol solution containing 0.1mM CaCL and incubated 2X.
The concentration was adjusted to 10S cells/ml. These protoplast solutions were mixed at a ratio of 1:1 and fused using an electric fusion device (high voltage pulse type fine &!7 fusion device R5G100O1 manufactured by Silvia Research Co., Ltd.). The high frequency is l
A DC pulse of 50 μsec and IKV/cm was applied twice (1 second interval) for 10 seconds at 70 V/cII.The fusion rate was approximately 5 to 10%.Next, the treated protoplast suspension was Transfer the suspension to a medium (2,
0pp+s NAA, 1. The protoplasts of Jiaogulan cultured cells did not divide in this 1/2 diluted medium and were cultured after about 1 month. The obtained colonies were subcultured while decreasing the concentration of mannitol in the medium, and many small calli were obtained. Among them, small calli that were morphologically different from calli derived from luffa cotyledon protogelasts were selected. When the selected small calli were analyzed in the same manner as in 1, they showed hybridity, and hybrid cells could be obtained.

Example 3 Preparation of Extract The hybrid loofah and Jiaogulan hybrid cells (F3) of Example 1, which had been subcultured for two years, were thoroughly washed with purified water and lyophilized. Add 101 parts of water to 100 g (dry weight) of the cells, and
After extraction at 5° C. for 3 hours and filtering off the residue, the filtrate was concentrated and then lyophilized to obtain the extract at 35°C. This extract of Tokuike was used in the following Examples. In addition, the amounts shown in the following Examples and Comparative Examples refer to parts by weight.

Example-4 Lotion formulation Amount A) Loofah and Jiaogulan hybrid cell extract 0.1 parts 1.3-
Butylene glycol 8.0 Glycerin 2.0 Xanthan gum
0.2 Purified water
45.1B) Ethanol
5.OP-methyl hydroxybenzoate
0.1 Polyoxyethylene (40) Hydrogenated castor oil 0.1 Fragrance Appropriate amount Edge degree water 10
．． 0 Manufacturing method; Component ^ and component B are each uniformly dissolved and mixed to form a product.

Comparative Example 4 Conventional lotion In Example 4, the hybrid cell extract of loofah and Jiaogulan was replaced with an extract of the loofah plant, and a conventional lotion was prepared. An extract of the loofah plant was prepared in the same manner as in Example 3 using 10 g of dried aerial parts of the loofah.

Example-5 Cream formulation Amount A) Stearic acid 4.0 parts Cetyl alcohol 3.0 Stearyl alcohol 1.0 Liquid paraffin
6.5 Vaseline
1O10 sorbitan monostearate
1.5 polyoxyethylene (25) monostearate 3.0 B)! , 3-butylene glycol 5.
0 Potassium hydroxide 0.1 Loofah and Jiaogulan hybrid cell extract 0.005 Methyl P-hydroxybenzoate 0.2 Purified water 64.7C) Fragrance
Appropriate amount production method: After heating and dissolving oil phase component A and water phase component B at 70 to 75°C, add component B to component A and emulsify, add and mix component C while cooling, and heat to 30°C. Cool it and use it as a product.

Example-6 Emulsion formulation Amount ^) Stearic acid Stearyl alcohol Liquid paraffin Glycerin monostearate Sorbitan monooleate Polyoxyethylene (10) Sorbitan monooleate B) Glycerin Loofah and Jiaogulan species H cell extract 0.01 0.2 77.4 Prepare the product in the same manner as in the appropriate amount.

Methyl P-hydroxybenzoate Purified water C) Fragrance manufacturing method: Example-5 Example-7 Bag formulation 1, glycerin 2.1.3-butylene glycol 3, methyl P-hydroxybenzoate 4, polyoxyethylene (40 ) Hydrogenated castor oil 2.0 parts 2.0 8.0! , 3 1.5 0.8 6.0 Blend amount 15.0 parts 10.0 0.2 0.5 5, Loofah and Jiaogulan hybrid cell extract 1.06,
Citric acid 0.17,
Sodium citrate 0.38, fragrance appropriate amount S, purified water
73.8 Manufacturing method:
Dissolve each component uniformly to form a product.

Example 8 Foundation formulation Ingredients: 1, loofah and Jiaogulan hybrid cell extract 0.5 parts 2, stearic acid 2.43, propylene glycol monostearate 2.04, cetostearyl alcohol 0.25, liquid lanolin 2. 06, Liquid paraffin 3.07, Isopropyl myristate 8.5g, Butyl P-oxybenzoate 0.19, Purified water
58.210, sodium carboxymethylcellulose 0.21, bentonite 0.52. Propylene glycol 4.03, triethanolamine 1.14, methyl P-oxybenzoate 0.25, titanium oxide
8.0!6. talc
4.017, colored pigments
5.0+8. fragrance
Proper amount Manufacturing method: Ingredients! , 9 is heated to 70°C, and component 11 is added to it to swell it well. Ingredients 12 to this
Component 1 is added and dissolved, and then Component 1 is added and dissolved.
3 and 14 to form an aqueous phase. Components 2 to 8 are dissolved by heating and kept at 80°C to form an oil phase. To the aqueous phase, add component 17 that has been thoroughly mixed and crushed through a pulverizer, stir with a homomixer and keep at 75°C, add the oil phase to this aqueous phase while stirring,
Cool and ingredients at 45℃! 8 is added, stirred and cooled, and then used as a product.

Example-9 Hair lotion formulation Amount A) Stearic acid 2.0 parts Stearyl alcohol 2.0 Liquid paraffin 8.0 Glycerin monostearate 1.3 Sorbitan monooleate 1
．． 5 Polyoxyethylene (lO) sorbitan monooleate
0.8 B) Loofah and Jiaogulan hybrid cell extract 0.2 Glycerin 6. OP-methyl oxybenzoate 0. ! purified water
78. lC) Fragrance
Proper amount production method: Dissolve the oil phase component ^ and water phase component B at 70 to 75°C, add component B to the components to emulsify, add component C during cooling, and mix.
The product is cooled to 30°C.

Example-10 Bath additive formulation Blend amount 1, Sodium sulfate 43.0 parts 2,
Sodium bicarbonate 50.03, loofah and Jiaogulan hybrid cell extract 5.04, borax 2.05, yellow No. 202 (+) appropriate amount 6, fragrance appropriate amount Manufacturing method:
After pulverizing component 3, each component is thoroughly mixed to form a product.

(Effects of the Invention) By using the method for producing hybrid cells of loofah and Jiaogulan according to the present invention, which is characterized by cell fusion of loofah protoplasts and Jiaogulan protoplasts, the hybrid cells can be obtained efficiently and reproducibly. be able to.

The loofah and Jiaogulan hybrid cells of the present invention proliferated better than the cultured loofah and Jiaogulan cells.

Furthermore, the hybrid cell extract was superior to loofah or Jiaogulan plant extracts, loofah or Jiaogulan cultured cell extracts, and loofah water in terms of cell proliferation activity for human epithelial cells.

In addition, the cosmetic composition of the present invention containing the extract of loofah and Jiaogulan hybrid cells as an active ingredient had a better skin-conditioning effect than conventional cosmetic compositions.

The effects of the present invention will be explained in more detail using the following experimental examples.

Experimental Example-1 A proliferation test was conducted using hybrid cells of loofah and Jiaogulan.9 Cultured cells of loofah and Jiaogulan were used as controls.

Hybrid cells of loofah and Jiaogulan were produced in the same manner as in Example-1, and cultured in cell suspension! Those that had been subcultured for a year were used. Cultured loofah cells are: 1. Oppm2.4-D,
The cotyledons were placed on MS medium containing 0.5 ppm kinetin and 02% casamino acids, and the callus was induced by cell suspension culture using the same liquid medium! Those that had been passaged for one year were used.

Cultured Jiaogulan cells were treated with 0.5 ppm picloram,
0. The culture using the callus which was induced by placing the stem section on MS medium containing lppm kinetin and 0.2% casamino acid was subcultured in cell suspension culture in the same liquid medium for one year.1. oppm N^^, 0.5ppm
Using MS' medium containing BAP and 0.2% casamino acids, the cells were cultured on a rotary shaker (90 r, p, m,) at 25° C. and under illumination (20001 ux) for 24 hours.

The conclusion: While the cultured cells of loofah or Jiaogulan hardly proliferated, the hybrid cells of loofah and Jiaogulan multiplied about 7 times after 10 days of culture, and produced 5.5 g (dry weight) per 11 days of culture. Obtained cells.

When NAA or IAA was used as auxin in the culture medium, cultured loofah or Jiaogulan cells hardly proliferated. Therefore, instead, we tried using 2,4-D and picloram, which have high auxin activity, which were used for callus induction, but the results were not as good as the proliferation of loofah and Jiaogulan hybrid cells.

/ When the hybrid cells of loofah and Jiaogulan obtained in Example-1 and Example-2 were similarly examined, their proliferation was superior to cultured cells of loofah or Jiaogulan.

Experimental Example-2 The cell proliferation activity on human epithelial cells was investigated using an extract of loofah and Jiaogulan hybrid cells.
The extract obtained in Example 3 was used in the experiment, and the Asahi control sample was prepared as follows. Cultured cells of loofah or Jiaogulan were prepared using tb2.4-D or tb2.4-D, respectively, as shown in Experimental Example 1. Culture was carried out in a medium containing picloram, and extraction was carried out in the same manner as in Example 3.Extracts of loofah or Jiaogulan plants were obtained from dried loofah or Jiaogulan plants (cultivated products) in Example-
Each was prepared using the extraction method shown in 3. Loofah water was collected by a conventional method and freeze-dried before use.

Cell proliferation activity for human epithelial cells was investigated as follows.Human epithelial cells had JTC-17 (XX
-male) cells, use Eagle to make JTC-17 cells 2x 104 cells/ml.
's Eagle' containing the sample suspended in MEM medium.
s MEM medium Add cell suspension 11 on medium 3.5
cIl(φ) plastic petri dish at 37℃, 5%C
After culturing under O□ conditions, the final concentration of the sample was IOμg.
/ml of culture medium- without any sample added was used as a control.

On the 4th day of culture, the culture medium was replaced, and on the 7th day of culture, the cells were detached and the number of cells was measured using a hemocytometer.The results were then tabulated.
Shown in 2.

Table 2 Cell proliferation activity test 11: JTC-17 cell culture 1170 [I cell count As shown in Table 2, the extract of hybrid cells of loofah and Jiaogulan is the same as the extract of loofah or Jiaogulan plant. The cell proliferation activity for human epithelial cells was superior to extracts of cultured cells of loofah or Jiaogulan and loofah water.

Regarding the loofah and Jiaogulan hybrid cells (F4, F5, F6) obtained in Example-1 and the H-species cells of Loofa and Jiaogulan obtained in Example-2, Examples were obtained from the cells passaged for two years. Extracts were prepared in the same manner as in 3-3, and their cell proliferation activity against human epithelial cells was examined. All of them were superior to the above-mentioned plant cultured cells and loofah water. When the cell proliferation activity of the ethanol extract against human epithelial cells was examined, it was found to be superior to the above-mentioned plant cultured cells and loofah water.

Experimental Example-3 In order to clarify the effect of the cosmetic of the present invention, 25 to 50
A usage test was conducted on 30 ordinary women of the same age.

A questionnaire survey regarding the skin conditioning effect was conducted using a double-blind method. A lotion containing a hybrid cell extract of loofah and Jiaogulan as an active ingredient (the lotion of Example-4) and a lotion containing a loofah plant extract as an active ingredient (Comparative Example-4) were prepared.
The results after using the product for several months are shown in Table 3. During the use test, not a single skin problem occurred.

Table 3 Questionnaire Results As is clear from Table 3, this is a lotion whose active ingredient is an extract of loofah and Jiaogulan hybrid cells! 9) It has a skin conditioning effect superior to conventional lotions containing loofah plant extract as an active ingredient. Conventional lotions were prepared in the same manner as in Example 4, and a comparative use test was conducted in the same manner as in Experimental Example 3, except that extracts of loofah and Jiaogulan hybrid cells were used as the active ingredients. The lotion had a better skin conditioning effect.

Hybrid cell of loofah and Jiaogulan F4 obtained in Example-1
, F5. A lotion containing F6 and the extract of loofah and Jiaogulan hybrid cells obtained in Example-2 as active ingredients was prepared in the same manner as in Example-4, and the above-mentioned conventional lotion was prepared in the same manner as in Experimental Example-3. When we conducted a comparative test with a lotion, we found that the lotion containing loofah and Jiaogulan hybrid cell extract as an active ingredient had better skin-conditioning effects.

Comparative use tests were similarly conducted for the cosmetics of Examples 5 to 10, and the cosmetics containing loofah and Jiaogulan hybrid cell extracts showed superior skin conditioning effects.

[Brief explanation of the drawing]

FIG. 1 is a photograph showing an electrophoretic band pattern in isozyme analysis of glucose-6-phosphate dehydrogenase. B shows callus derived from loofah cotyledon protoplasts, and G shows cultured cells of Jiaogulan. Also. Mix is a mixture of B and G. Fl, F
2, F3, F4. F5 and F6 indicate callus derived from the single plant obtained in Example-1. FIG. 2 is a schematic diagram of the electrophoretic band pattern in the isozyme analysis of glucose-6-phosphate dehydrogenase shown in FIG. 1. The symbols are the same as in Figure-1. Figure-1 Figure-2

Claims (1)

[Scope of Claims] 1. Hybrid cells of loofah and Jiaogulan obtained by cell fusion of loofah protoplasts and Jiaogulan protoplasts. 2. A method of action of a loofah and Jiaogulan hybrid cell, which is characterized by cell fusion of loofah protoplasts and Jiaogulan protoplasts. 3. A cosmetic containing an extract of hybrid cells of loofah and Jiaogulan as an active ingredient.