AU2018100588A4

AU2018100588A4 - Cell culture mediums containing aqueous extract derived from herbal medicines

Info

Publication number: AU2018100588A4
Application number: AU2018100588A
Authority: AU
Inventors: Mei-Yue Huang
Original assignee: MARIA VON MED BIOTECHNOLOGY CO Ltd
Current assignee: MARIA VON MED-BIOTECHNOLOGY Co Ltd
Priority date: 2018-05-09
Filing date: 2018-05-09
Publication date: 2018-06-07
Anticipated expiration: 2026-05-09

Abstract

The present invention relates to new uses of the aqueous extracts derived from Hedysarum polybotrys Hand.-Mazz., Alisma plantago-aquatica (Sam.) 5 Juzep, Momordica charantia L., Eisenia foetida and the combinations thereof in cell culture. The aqueous extracts disclosed herein is especially useful for substituting animal serum in a culture medium, so that the content of animal serum in the cell culture medium can be reduced greatly, thereby reducing the production cost of cells and reducing the health risk resulted from animal serum.

Description

The present invention relates to new uses of the aqueous extracts derived from Hedysarum polybotrys Hand.-Mazz., Alisma plantago-aquatica (Sam.)

Juzep, Momordica charantia L., Eisenia foetida and the combinations thereof in cell culture. The aqueous extracts disclosed herein is especially useful for substituting animal serum in a culture medium, so that the content of animal serum in the cell culture medium can be reduced greatly, thereby reducing the production cost of cells and reducing the health risk resulted from animal serum.

2018100588 09 May 2018

CELL CULTURE MEDIUMS CONTAINING AQUEOUS EXTRACT DERIVED FROM HERBAL MEDICINES

FIELD OF THE INVENTION

The present invention relates to aqueous extracts derived from herbal medicines, and more particularly, to new compositions of the aqueous extracts derived from a single herbal medicine, and the combined aqueous extracts derived from two or more herbal medicines, as well as the uses thereof in cell culture.

BACKGROUND OF INVENTION

Fibroblasts exist in many tissues of animal body, for producing extracellular matrix and collagen. They are responsible for maintaining the basic structure of connective tissues and play a leading part in wound healing.

Cultured fibroblasts exhibit the effects of enhancing tissue repair and are suitable for a wide range of applications, including acute and chronic wound healing, plastic surgery and reconstruction operation. In terms of wound healing, for example, fibroblasts may be cultured in a biodegradable scaffold to constitute a skin substitute. However, it is questionable whether such products would stand up to the scrutiny for safety and quality inspection. For instance, the fibroblast preparations should be subject to strict scrutiny as to whether the cells are derived from human tissues, whether the preparations are produced using aseptic processes, whether there is contamination, whether the desired activities are maintained after freezing, and whether there is a risk of rejection or virus infection under the circumstance that the cells are derived from a non-autologous source. In addition, how to minimize the cost and to maximize the product usability are the urgent topics the persons of

2018100588 09 May 2018 related technical fields want to solve.

In the present medical cosmetology, autologous fibroblasts may be applied to human body as tissue filler, replacing the conventional injection of bovine collagen which may have a risk of rejection or infection. In order for application of fibroblasts in autologous injection, it is imperative to reduce the amount of fetal bovine serum (FBS) used in fibroblast culture without compromising the effectiveness of culturing human fibroblasts. At present, fibroblasts are typically cultured in DMEM supplemented with 10% FBS, wherein FBS provides the nutrients and growth factors for fibroblast growth.

However, some components of FBS may enter the fibroblasts during cell culture, raising safety concerns about the risks of rejection, allergy and even disease transmission. Therefore, there is a need for a FBS replacement or cell culture supplement which can facilitate the growth of fibroblasts.

SUMMARY OF THE INVENTION

The dermal fibroblasts specialize in producing extracellular matrices, such as collagen and elastin, so as to maintain the elasticity and density of skin. Under the circumstances that the dermal layer has sufficient fibroblasts to secrete collagen, skin wrinkles may be smoothed and skin elasticity may be maintained.

Therefore, the quantity and viability of dermal fibroblasts are closely bound up with the health, moisture and compactness of skin. The present inventor established an in vitro model of primary human dermal fibroblast cell culture adapted for screening pharmaceutical ingredients which can enhance the viability of fibroblasts, and/or facilitate secretion collagen and/or growth factors from fibroblasts, and/or enhance the antioxidant activity of fibroblasts, and which may be useful in manufacture of healthcare products to achieve anti-wrinkle and anti-aging effects.

2018100588 09 May 2018

By using the in vitro model to test simple or combined extracts derived from certain Chinese herbal medicines, the inventor note that the aqueous extracts derived from Hedysarum polybotrys Hand.-Mazz., Alisma plantago-aquatica (Sam.) Juzep, Momordica charantia L. and Eisenia foetida, either used alone or in combination, can facilitate the growth of human or animal cells, especially fibroblasts, such as human dermal fibroblasts, under an in vitro culture condition containing a low content of FBS. Therefore, said aqueous extracts are useful in reducing the amount of FBS used in cell culture medium, thereby reducing the production cost of animal cells. Especially, when introduced into a human patient, the fibroblasts produced according to this application offer a reduced health risk to the human patient.

Accordingly, in the first aspect provided herein is a cell culture medium supplement comprising:

an aqueous extract derived from one or more biological species selected from the group consisting of Hedysarum polybotrys Hand.-Mazz., Alisma plantago-aquatica (Sam.) Juzep. Momordica charantia L., Eissenia foetida and combinations thereof.

In the second aspect provided herein is a cell culture medium comprising: the aqueous extract described above; and a basal medium supplemented with animal serum at a concentration of substantially less than 10% by volume based on the total volume of the cell culture medium.

DETAILED DESCRIPTION OF THE INVENTION

Fig. 1 is a histogram showing the cell viability of fibroblasts using extracts derived from individual herbal medicines to culture the fibroblasts for 3 days;

Fig. 2 is a histogram showing the cell viability of fibroblasts using extracts

2018100588 09 May 2018 derived from individual herbal medicines to culture the fibroblasts for 6 days;

Fig. 3 is a histogram showing the amounts of collagen secreted from fibroblasts using extracts derived from individual herbal medicines to culture the fibroblasts for 3 days;

Fig. 4 is a histogram showing the amounts of collagen secreted from fibroblasts using extracts derived from individual herbal medicines to culture the fibroblasts for 6 days;

Fig. 5 is a histogram showing the amounts of vascular endothelial growth factor (VEGF) secreted from fibroblasts using extracts derived from individual herbal medicines to culture the fibroblasts for 3 days;

Fig. 6 is a histogram showing the amounts of VEGF secreted from fibroblasts using extracts derived from individual herbal medicines to culture the fibroblasts for 6 days;

Fig. 7 is a histogram showing the cell viability of fibroblasts using combined extracts derived from two or more herbal medicines to culture the fibroblasts for 3 days;

Fig. 8 is a histogram showing the cell viability of fibroblasts using combined extracts derived from two or more herbal medicines to culture the fibroblasts for 6 days;

Fig. 9 is a histogram showing the amounts of collagen secreted from fibroblasts using combined extracts derived from two or more herbal medicines to culture the fibroblasts for 3 days;

Fig. 10 is a histogram showing the amounts of collagen secreted from fibroblasts using combined extracts derived from two or more herbal medicines to culture the fibroblasts for 6 days;

Fig. 11 is a histogram showing the amounts of VEGF secreted from fibroblasts using combined extracts derived from two or more herbal medicines

2018100588 09 May 2018 to culture the fibroblasts for 3 days;

Fig. 12 is a histogram showing the amounts of VEGF secreted from fibroblasts using combined extracts derived from two or more herbal medicines to culture the fibroblasts for 6 days;

Fig. 13 are fluorescence microscope images showing the fibroblasts that express FSP-1 after treatment with individual extracts derived from herbal medicines; and

Fig. 14 is a histogram showing the percentages of the fibroblasts that express FSP-1 after treatment with individual or combined extracts derived from herbal medicines.

DETAILED DESCRIPTION OF THE INVENTION

This application relates generally to use of traditional herbal medicines, including H. polybotrys, A. plantago-aquatica, M. charantia and E. foetida, in cell culture and skin care.

As used herein, the term Hedysarum polybotrys Hand.-Mazz., or abbreviated as ”H. polybotrys,” encompasses the newly harvested, unprocessed and processed whole plant and plant parts of the botanical species, especially the underground part thereof, such as the dried roots thereof commercially available from the traditional herbal medicine distributors.

The term Alisma plantago-aquatica (Sam.) Juzep, or abbreviated as A. plantago-aquatica,” as used herein encompasses the newly harvested, unprocessed and processed whole plant and plant parts of the botanical species, especially the underground part thereof, such as the dried rhizomes thereof commercially available from the traditional herbal medicine distributors. Normally, the rhizome parts were picked in winter when the stem and leaf parts began to wither. The rhizome parts were washed, roasted to dry, trimmed to

2018100588 09 May 2018 remove fibrous roots, re-wetted with water, and then sliced and dried under the sun. In traditional herbal medicine distributors, A. plantago-aquatica is typically sold in the form of dried rhizomes processed by stir-frying with bran or stir-frying with salty water.

The term Momordica charantia L. or abbreviated as M. charantia,” as used herein encompasses the newly harvested, unprocessed and processed whole plant and plant parts of the botanical species, especially the fruit parts thereof.

The term Eisenia foetida, or abbreviated as E. foetida, as used herein encompasses the newly harvested, unprocessed and processed whole worms or worm parts of the animal species, such as the unprocessed whole worms sold as fish bait in fishing tackle shops, or the processed whole worms or worm segments sold in traditional Chinese medicine distributors. In traditional processing, E. foetida is usually caught in summer and autumn, eviscerated and washed before solarization or cold drying. The dried worm bodies are used as medicine directly or after being stir-fried with yellow wine.

The term “aqueous extract,” as used herein, may refer to a composition prepared by contacting one or more traditional medicines with an aqueous solvent following standard extraction procedures. The term “derived from” as used herein is intended to indicate the traditional medicine(s) from which the aqueous extract is prepared. Non-limiting examples of the aqueous solvent include water, Ci-6 alkanols, Cm alkylene glycols, aqueous carbohydrate solutions, salt solutions thereof, and mixtures thereof. Desirably, the aqueous solvent is selected from water and salt solutions thereof. The traditional medicine(s) may be ground beforehand, and the pressure and temperature during the extraction procedures may be elevated for enhancing the extraction efficiency. In a preferred embodiment, the aqueous extract is obtained by extraction with deionized water at a high temperature, such as at about 100°C under ambient

2018100588 09 May 2018 pressure. The duration of extraction depends on the yield of extraction and normally lasts 30 minutes to one day, preferably 1-12 hours, such as 1-3 hours.

The term “aqueous extract” encompasses crude extracts, prepared by a simple aqueous extraction, as well as crude extracts that have been subjected to one or more separation and/or purification steps, including substantially purified and purified active ingredient(s) and concentrates or fractions derived from a crude extract by subjecting the crude extract to one or more additional extraction, concentration, fractionation, filtration, distillation or other purification step. The extract may be in liquid form, such as a solution, concentrate or distillate, or in semi-liquid form, such as a gel. The aqueous extract can, if appropriate and desired, be lyophilized and stored in a sterile ampoule ready for reconstitution by the addition of an aqueous solvent, such as sterile water.

According to this application, the individual aqueous extracts derived from H. polybotrys, A. plantago-aquatica, M. charantia and E. foetida, as well as the combinations thereof, can enhance the growth of human or animal cells, especially fibroblasts, such as human dermal fibroblasts, at a low content of FBS, indicating that the aqueous extracts may be used as a supplement in animal cell culture media, so that the amount of animal serum in the media may be reduced. In general, the animal cell culture media are supplemented with 10% to 20% animal serum, so as to provide the proteins, inorganic salts, growth factors, hormones and attachment factors for cell growth. The term animal serum may refer to liquid products derived from non-human animal blood, wherein the blood cells, fibrinogen and fibrin are removed. The animal serum is suitable for addition to a culture medium to provide nutrients needed for cell growth. The animal serum includes, but is not limited to, FBS, newborn calf serum, calf serum and other animal serums, such as the serum products derived from horse and camel.

2018100588 09 May 2018

This application uses primary human dermal fibroblasts as a platform for screening aqueous extracts derived from herbal medicines. As shown in

Examples 1 and 7 below, in comparison to the control group using a basal medium supplemented with 0.5% FBS, the basal media contain, in addition to

0.5% FBS, individual or combined aqueous extracts derived from H. polybotrys,

A. plantago-aquatica, M. charantia and E. foetida can increase the cell numbers of fibroblasts by 1.5 times and 1.4-2.4 times on Day 3 and Day 6 of culture, respectively, suggesting that said aqueous extracts can enhance the growth of fibroblasts. Example 7 further shows that the individual and combined aqueous extracts disclosed herein, in combination with 0.5% FBS, may achieve a cell proliferation rate of 50-66.7% as compared with that obtained using a culture medium supplemented with a conventional amount of FBS (10%). Without wishing to be bound by theory, it is believed that said aqueous extracts not only contain the nutrients needed by animal cells, but also include certain components which may stimulate fibroblasts to secrete growth factors, thereby enhancing cell proliferation. Example 12 also evidences that the fibroblasts treated with the aqueous extracts disclosed herein will neither change their characteristics nor differentiate to mature cells.

It is worthwhile to note that, as shown in Example 7, when a combined aqueous extract derived from H. polybotrys and E. foetida, a combined aqueous extract derived from H. polybotrys, A. plantago-aquatica and M. charantia, a combined aqueous extract derived from A. plantago-aquatica, M. charantia and E. foetida, a combined aqueous extract derived from H. polybotrys, M. charantia and E. foetida, and a combined aqueous extract derived from H. polybotrys, A.

plantago-aquatica, M. charantia and E. foetida are used to culture fibroblasts, the cells can be proliferated over two times, and these combined extracts exhibit higher cell proliferation effects as compared with individual extracts alone.

2018100588 09 May 2018

Without wishing to be bound by theory, it is believed that the constituents in the five combined extracts provide synergistic effects on cell proliferation. In a more preferred embodiment, the aqueous extract is selected from a combined aqueous extract derived from H. polybotrys and E. foetida.

Accordingly, this application provides a cell culture medium supplement, which comprises an aqueous extract derived from one or more biological species. The biological species is selected from the group consisting of H. polybotrys, A. plantago-aquatica, M. charantia and E. foetida. The term cell culture medium supplement may refer to a composition adapted for being added to a cell culture medium to assist in maintaining the viability of cells inoculated in the culture medium and/or enhancing the proliferation of the cells. The culture medium supplement is adapted to support human or animal cell cultures, preferably supporting human or animal fibroblast cultures, such as supporting human dermal fibroblast cultures. Since the functions of the animal serum in a culture medium can be substituted in part or completely by the aqueous extracts disclosed herein, the content of the animal serum in the cell culture medium can be reduced greatly from the conventional content of 10% to an amount of substantially less than 10% by volume, preferably an amount of substantially less than 5% by volume, more preferably an amount of substantially less than 1% by volume, such as an amount of about 0.5% by volume, based on the total volume of the cell culture medium.

Therefore, this application further contemplates a cell culture medium comprising the cell culture medium supplement described above, and a basal medium supplemented with animal serum at a concentration of substantially less than 10% by volume, preferably at a concentration of substantially less than 5% by volume, and more preferably at a concentration of substantially less than 1% by volume, such as at a concentration of about 0.5% by volume, based on the

2018100588 09 May 2018 total volume of the cell culture medium.

The term “basal culture medium” as used herein may refer to any serum-free liquid culture medium containing inorganic salts, amino acids and vitamins usually required to support growth of human or animal cells in vitro.

In some preferred embodiments, the basal culture medium is growth factor-free. Examples of the basal culture medium include but are not limited to Basal Medium Eagles (BME), Minimum Essential Medium (MEM), Dulbecco’s Modified Eagle's Medium (DMEM), Nutrient Mixture F-10 (HAM's F-10) and Nutrient Mixture F-12 (HAM's F-12). In a more preferred embodiment, the basal culture medium is DMEM.

The following embodiments are used only for illustration, not to limit the scope of the present invention. The experimental results in various embodiments are compared with the control group by Student's t test, #: 0.05 < p < 0.1; *: p < 0.05.

Preparative Example 1: Primary Culture of Human Dermal Fibroblasts

A skin specimen was obtained with donor's consent. The skin specimen was cut into 0.5 to 1 mm³ pieces. Afterwards, the skin pieces were placed in DMEM supplemented with FBS, collagenase and dispase, incubated at 37 °C for 16-18 hours, and centrifuged at 1,500 rpm for 5 minutes. The supernatant was removed, and the cells and skin pieces were scattered with FBS-containing DMEM and then dispensed in a flask uniformly and incubated at 37°C. The cells were passaged when reached 80% confluence and used in the following Examples between passage 3 and 6.

Preparative Example 2: Preparation of Aqueous Extracts

H. polybotrys and A. plantago-aquatica viqxq purchased from traditional herbal medicine distributors. E. foetida was purchased from a local fishing tackle shop. M. charantia was purchased from a local fruit and vegetable

2018100588 09 May 2018 market.

lOOg of H. polybotrys was added into 500g double distilled water and boiled at ambient pressure for 2 hours to generate a suspension of H. polybotrys weighted 210g.

lOOg of A. plantago-aquatica was added into 500g double distilled water and boiled at ambient pressure for 2 hours to generate a suspension of A. plantago-aquatica weighed 242g.

lOOg of M. charantia fruit was added into 500g 500g double distilled water and boiled at ambient pressure for 2 hours to generate a suspension of M.

charantia weighed 150g.

lOOg of unprocessed E. foetida was added into 500g double distilled water and boiled at ambient pressure for 2 hours to generate a suspension of E. foetida weighed 170g.

The suspensions were filtered through gauze respectively to remove debris. 15 The respective filtrates were collected and centrifuged at 6000 rpm for 30 minutes. The respective supernatants were collected and passed through a 0.2 pm filter. The resultants filtrates were collected as the stock extracts and stored at

-20 °C.

In the following Examples, the stock extracts were diluted several fold with 20 the culture medium used in the control group, and the cells were treatment by the diluted extracts. For example, H. polybotrys 100X refers to the diluted extract obtained by diluting the stock solution of H. polybotrys 100-fold with the culture medium used in the control group.

Example 1: Treatment of Cells with Individual Extracts

The fibroblasts prepared in Preparative Example 1 was inoculated in a

96-well culture plate and cultured in DMEM (Sigma Chemical Co., St. Louis, MO, USA) supplemented with 0.5% FBS (Gibco Co., NY, USA) in the presence ll

2018100588 09 May 2018 of the respective extracts prepared in Preparative Example 2 for 3 to 6 days. The control group was the fibroblasts of Preparative Example 1 cultured in DMEM supplemented with either 0.5% or E0% FBS. The supernatants and the cells were harvested for subsequent analyses.

Example 2: Enhancement Effects of Individual Extracts on Cell Proliferation

A commercially available XTT analysis kit (Sigma Chemical Co., St. Louis City, MO, USA) was used to treat the cells obtained in Example 1 according to manufacturer’ s instructions. The O.D. readings of respective wells in the culture plate were measured at 450 nm to obtain the cell viability. Figs. 1 and 2 show that the fibroblasts incubated with individual extracts derived from H. polybotrys, A. plantago-aquatica or E. foetida for either 3 or 6 days exhibited a significant increase in cell number as comparison to the control group which was incubated with 0.5% FBS alone. The proliferation rates of the tested groups on Day 3 were about 1.2 times compared with the control group which was incubated with 0.5% FBS alone, whereas the proliferation rates of the tested groups on Day 6 exhibited an increase of more than 1.5 times compared with the control group. In the case where the fibroblasts were treated with the extract derived from M. charantia, the cell proliferation was not enhanced until Day 6. This example indicates that the four extracts disclosed herein have the capability of enhancing the proliferation of human dermal fibroblasts.

Example 3: Effects of Individual Extracts on Collagen Secretion from Fibroblasts

A commercially available human recombinant collagen Type I detection kit (Gibco Co., NY, USA; kit No. PRO-479A) was used to treat the supernatants obtained in Example 1 according to manufacturer’ s instructions, so that the amounts of collagen secreted from the fibroblasts were obtained.

Fig. 3 shows the amounts of collagen secreted from fibroblasts by using the individual extracts derived from herbal medicines to culture the fibroblasts for 3

2018100588 09 May 2018 days. In comparison to the control group which was incubated with 0.5% FBS alone, the extract derived from H. polybotrys significantly facilitated collagen secretion at three tested concentrations, and the 200X diluted extract exhibited the best effects. The extract derived from A. plantago-aquatica, at 4800X dilution, enhanced collagen secretion significantly. The extract derived from E. foetida, at 4000X and 2000X dilution, stimulated the fibroblasts to secrete collagen significantly. However, the extract derived from M. charantia failed to stimulate collagen secretion at the tested concentrations. Fig.4 shows the amounts of collagen secreted from fibroblasts by using the individual extracts to culture the fibroblasts for 6 days. Both of H. polybotrys extract at 200X dilution and E. foetida extract at 8000X dilution enhanced collagen secretion significantly. This example shows that the extracts derived from H. polybotrys or E. foetida, in combination with 0.5% FBS, stimulated fibroblasts to secrete collagen, and such stimulation effects were superior to that achieved by using

1.0% FBS alone.

Example 4: Effects of Individual Extracts on Secretion of Growth Factors from

Fibroblasts

A commercially available VEGF detection kit (R&D Systems Co., MO, USA; VEGF Duoset® EFISA Kit, No. DY293B) was used to treat the supernatants obtained in Example 1 according to manufacturer’ s instructions, so that the amounts of VEGF secreted from the fibroblasts were obtained.

Figs. 5 and 6 show the amounts of VEGF secreted from fibroblasts by using the individual extracts derived from herbal medicines to culture the fibroblasts for 3 and 6 days. There is no significant difference between the 0.5% FBS control group and the individual extracts in terms of the VEGF secretion.

According to Examples 1 to 4 where fibroblasts were cultured in the presence of the individual extracts disclosed herein, the H. polybotrys extract at

2018100588 09 May 2018

400X dilution, the E. foetida extract at 2000X dilution, the A. plantago-aquatica extract at 4800X dilution, and the M. charantia extract at 2500X dilution, exhibited the highest effects. As such, the diluted extracts of H. polybotrys 400X,

E. foetida 2000X, A. plantago-aquatica 4800X and M. charantia 2500X are used in examples below.

Example 5: Treatment of Cells with Combined Extracts

The procedure of Example 1 was repeated, except that the diluted extracts of

H. polybotrys 400X, E. foetida 2000X, A. plantago-aquatica 4800X and M. charantia 2500X were used, either individually or in combination, for culturing fibroblasts for 3 to 6 days. The control groups were the fibroblasts of Preparative Example 1 cultured in DMEM supplemented with 0.5% or 1.0% FBS in the absence of the extracts disclosed herein. The supernatants and cells were collected and analyzed separately.

Example 6: Enhancement Effects of Combined Extracts on Cell Proliferation

A commercially available XTT analysis kit (Sigma Chemical Co., St. Louis

City, MO, USA) was used to treat the cells obtained in Example 5 according to manufacturer’ s instructions. The O.D. readings of respective wells in the culture plate were measured at 450 nm to obtain the cell viability. Figs. 7 and 8 show that the fibroblasts incubated with either the individual extracts or the combined extracts derived from H. polybotrys, A. plantago-aquatica and E. foetida for either 3 or 6 days exhibited a significant increase in cell number as comparison to the control groups. The proliferation rates of the tested groups on Day 3 were about 1.5 times compared with the control groups, whereas the proliferation rates of the tested groups on Day 6 exhibited an increase of more than 2 times compared with the control groups. This example further shows that the enhancement actions of the four diluted extracts did not interfere with one another. It is worthwhile to note that as shown in Fig. 8, the combined extracts of

2018100588 09 May 2018

H. polybotrys+E. foetida, H. polybotrys+A. plantago- aquatica+M. charantia, A.

plantago-aquatica+M. charantia+E. foetida, H. polybotrys+M. charantia+E.

foetida, and H. polybotrys+A. plantago-aquatica+M. charantia+E. foetida, led to an increase in cell proliferation rate of more than 2 folds, and the combined extracts exhibited higher effects on cell proliferation as compared to individual extracts alone, he individual and combined extracts disclosed herein, in combination with 0.5% FBS, achieved about 50% to 66.7% stimulatory effects relative to the control groups using 5% and 10% FBS.

Example 7: Effects of Combined Extracts on Collagen Secretion from Fibroblasts

A commercially available human recombinant collagen Type I detection kit (Gibco Co., NY, USA; kit No. PRO-479A) was used to treat the supernatants obtained in Example 5 according to manufacturer’ s instructions, so that the amounts of collagen secreted from the fibroblasts were obtained.

Figs. 9 and 10 show the amounts of collagen secreted from fibroblasts by using the individual and combined extracts disclosed herein to culture the fibroblasts for 3 or 6 days. Compared to the control group using 0.5% FBS, the combined extract of H. polybotrys and E. foetida led to an increase in collagen secretion by nearly 3 folds, and the enhancement effect thereof was much higher than that achieved by 1.0% FBS.

Example 8: Effects of Combined Extracts on Secretion of Growth Factors from

Fibroblasts

A commercially available VEGF detection kit (R&D Systems Co., MO, USA; VEGF Duoset® ELISA Kit, No. DY293B) was used to treat the supernatants obtained in Example 5 according to manufacturer’ s instructions, so that the amounts of VEGF secreted from the fibroblasts were obtained.

Figs. 11 and 12 show the amounts of VEGF secreted from fibroblasts by using the individual and combined extracts disclosed herein to culture the

2018100588 09 May 2018 fibroblasts for 3 or 6 days. There is no significant difference between the 0.5%

FBS control group and the individual extracts in terms of the VEGF secretion.

Example 9: Effects of Extracts on Fibroblast Characteristics

In order to ensure that the extracts disclosed herein will not change the properties of fibroblasts, this example used antibody to stain the fibroblasts obtained in Example 5, to see if the fibroblast specific protein 1 (FSP-1) is still expressed on the cells. As shown in Fig. 13, the fibroblasts incubated with the individual extracts disclosed herein for 3 day can express FSP-1. Fig. 14 shows that the percentages of FSP-1 expressed on the fibroblasts treated with the individual or combined extracts disclosed herein, which represent the purity of the fibroblasts. The results show that the percentages of the fibroblasts that expressed FSP-1 in the respective tested groups were over 95%, indicating that the individual or combined extracts disclosed herein will not affect the characteristics of primary dermal fibroblasts.

2018100588 09 May 2018

Claims

CLAIMS:

1. A cell culture medium comprising:

a cell culture medium supplement, comprising an aqueous extract derived from one or more biological species selected from the group consisting of

5 Hedysarum polybotrys Hand.-Mazz., Alisma plantago-aquatica (Sam.) Juzep. Momordica charantia L., Eissenia foetida and combinations thereof; and a basal medium supplemented with animal serum at a concentration of substantially less than 10% by volume based on the total volume of the cell culture medium.
2. The cell culture medium according to claim 1, wherein the aqueous extract is selected from the group consisting of a combined aqueous extract derived from H. polybotrys and E. foetida, a combined aqueous extract derived from H. polybotrys, A. plantago-aquatica and M. charantia, a combined aqueous

15 extract derived from A. plantago-aquatica, M. charantia and E. foetida, a combined aqueous extract derived from H. polybotrys, M. charantia and E. foetida, and a combined aqueous extract derived from H. polybotrys, A. plantago-aquatica, M. charantia and E. foetida.

20
3. The cell culture medium according to anyone of the preceding claims, wherein the aqueous extract is selected from a combined aqueous extract derived from H. polybotrys and E. foetida.
4. The cell culture medium according to anyone of the preceding claims,

25 wherein the basal medium is supplemented with animal serum at a concentration of substantially less than 5% by volume based on the total volume of the cell culture medium.
5. The cell culture medium according to anyone of the preceding claims, wherein the basal medium is supplemented with animal serum at a concentration of substantially less than 1% by volume based on the total volume of the cell

5 culture medium.

2018100588 09 May 2018

2018100588 09 May 2018

FIGURES

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Coitagen (ng/ml)

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Cell Viability [%)

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0.5% FBS

10% FBS

5% FBS

10% FBS

H. polybotrys ΑΟΰΧ.

A. plantago-aquatica 480QX

M. charantia2500X £/oeiwa2000X

Cell Viability (%)

Kj Kj LaJ Lw O vn O cjn O O O O <ra‘

V

H. polybotrys + A plantago-aquatica H. polybotrys+/ML charantia

H. polybotiys+Efoetida

A. plantago-aquatica+M. charantia

A plantago-aquatica+Efoetida

M. charantia + E foetida

H. polybotrys+A plantago-aquatica+ M, charantia

H. polybotrys+A plantago-aquatica+ Efoetida

A plantago-aquatica+M. charantia+ Efoetida

K pdybotrys+M. charantia+

Efoetida

H. polybotrys+A. plantago-aquatica +

M charantia + Efoetida

2018100588 09 May 2018

TO*

V kO

0.5% FBS

1.0% FBS

H. polybotrys QVGX.

A pfantog0-aguafra48OQX

M. charantia 2330X

E foetida 21XX0i

H. pofybottys+A pfarfiago-aquatica

H. polybotrys+M. charantia

H. polybotrys + E foetida

A plantago-aquatica+M. charantia

A plantago-aquadcxi+E foetida

M. charantia+£ foetida

H. polybotrys+A plantago-aquatica+ M. charantia

H. polybotrys+A plantago-aquatica+ £ foetida

A plantagooquatiat+M. charantia+ E foetida

H. polybotrys+M. charantia + £ foetida

H. polybotrys+A plantago-aquatica+ M. charantia + E foetida

Type 1 Collagen (ng/ml)

9/14

2018100588 09 May 2018

Type 1 Collagen (ng/ml) era

I-¹ o

05% FBS

L0%FBS

5%FBS

10% FBS

H. polybotrys 400X

A plantago-aquatica 48IXSi

M.dwtantia25(XM

EjoetidaXXXM.

H. pa/jflbotryr+A plantago-aqucrtica H. polybotrys-t-M. charantia

H. polybotrys+£ foetida

A plantago-aquatica+M. charantia

A plantago-ciqucrtica+E foetida

M. charantia+E foetida

H. polybotrys+A plantago-aquatica+ M. charantia

H. pofybotrys+A plantago-aquatica+ £ foetida

A plantago-aquatica+M. charantia+ £ foetida

H. polybotrys+M. charantia+ £ foetida

H. polybotrys+A plantago-aquatica +

M. charantia+E foetida

Ii

10A4

2018100588 09 May 2018

0.5%FBS

1.0%FBS

HpoiybotrysAOOX.

A.pkintagcxaquatica49Xiy

M.charantia 2500X

EfixtidaXKXlX

H.pofybotrys+A plantago-aquatica

H.polybotrys+ M. chararrtia

H. pofybotrys+Efoedda

A plantago-aquatica+M. charanda

A plantago-aquatica+£ foetida

M. chamnda +£ foetida

H. pofybotrys+A plantago-aquatica+ M.drarantia

H. pofybotrys+A p/artago-aquatica+ E foetida

Aplantag&aquatka+M. charnnda+ E foetida

H pofybotrys+M, charanda+ Efoedda

H. pofybotrys+A pktntago-c/quatica+ M. charanda +E foetida

11/14

2018100588 09 May 2018

05%FBS

10% FBS

5% FBS

10% FBS

H. pofybotrys 400X

A pfantugo-aquatiai 4S00X

M. charantia 2500Χ

EJoetKtoZOOQX — H. pofybotrys+A pfantago-aquatxa

fsj H. pofybotrys + M. charantia fi pofybotrys+E foetida

A pfantago-aquatxa+M. charantia

A plantago-aqua&a+E foetida

M. charantia+E foetida

H. pofybotrys+A plantago-aqua&a+ M. charantia

H. pofybotrys+A plantago-aquatka+ Efoetida

A pfantago-aquatica+M. charantia+ Efoetida

H. pofybotrys+M. charantia+

Efoetida

H. pofybotrys+A plantago-aquatka+

M. charantia+E foetida

VEGF (pg/mlj kj -t· to o

Ο ο Ϊ? O ft o ι?τ#τ

2018100588 09 May 2018

U1

I ? 9 CTO νιΛτ

2018100588 09 May 2018

05% FBS

L0%FBS

H. polybotrys 4OOX

Aplantagoaquatica4900X

M. charantia 250GX

E/oetfda 2OOOX

H. pofybatrys+A. plantago-aquatica

H. polybotrys+M. charantia

H. polybotrys+E foetida

A plantago-aquatica+M. charantia

A pfantago-aquatica+£ foetida

M. charantia+E foetida

H. polybattys+A plantago-aquatica + M. charantia

H. polybattys+A plantago-aquatica + E foetida

A plantago-aquatica+M. charantia+ E foetida

H. pofybotrys+M. charantia + Efoetida

H. polybotrys+A plantago-aquatica + M. charantia+E foetida