WO2021133309A1

WO2021133309A1 - Red californian worm extract and the use thereof

Info

Publication number: WO2021133309A1
Application number: PCT/TR2020/051204
Authority: WO
Inventors: Ilkay Erdogan Orhan; Turan YUKSEK; Aysegul MENDI; Fatma Sezer SENOL DENIZ; Melis Sirin ASLAN
Original assignee: Gazi Universitesi
Priority date: 2019-12-26
Filing date: 2020-12-01
Publication date: 2021-07-01
Also published as: TR201921760A1

Abstract

It has been seen that the extracts obtained from the liquid fertilizers of Eisenia foetida by the lyophilization and from the solid fertilizers or from the worm itself by the ethanol extraction have the inhibitory effect against the tyrosinase, elastase, collagenase, xanthine oxidase, and cholinesterase enzymes, and increase the calcium and osteocalcin concentration for the ossification of the dental pulp mesenchymal stem cells and the collagen concentration in the extracellular matrix. According to the results obtained, it is possible to use the Eisenia foetida extract in the products that provide the ossification and cell sternness in dental and in the anti-aging cosmetic products.

Description

RED CALIFORNIAN WORM EXTRACT AND THE USE THEREOF

Technical Field

The present invention relates to the Red Californian Worm (Eisenia foetida) extract, the method for preparing this extract, the pharmaceutical compositions comprising this extract, and the use of the extract and compositions.

Prior Art

Eisenia foetida (or Eisenia fetida), known as the Red Californian Worm, is a species of the culture worms, and the solid and liquid biological fertilizers obtained from these worms which feed on the different types of the organic wastes are used especially in the organic agriculture today.

In the state of the art, there is no cosmetic or regenerative composition comprising the extracts prepared from Eisenia foetida, as well as there are very few pharmacological studies on the extracts. However, there is no study on the biological activity and chemical composition of the Eisenia foetida extracts yet.

Summary of the Invention

There are limited number of studies in terms of Pharmacognosy on the extracts obtained by the homogenization of Eisenia foetida species which is cultured due to its rapid growth characteristic and used as a biological fertilizer. Therefore, as a result of the examination of the enzyme inhibitory effects of the solid and liquid fertilizers obtained from Eisenia foetida and of the worm itself and their biological activities on the mesenchymal stem cells, based on the worm extract, the extracts have been obtained, which are the natural raw materials to be used in the preparation of the different product formulations such as the cosmetic (anti-aging) products and different carrier systems (nanoparticle, liposome, etc.) that can be used in dentistry. With this research which constitutes the first study in the scientific literature, the Eisenia foetida extract which is easy-to-maintain and is a sustainable resource will function as a new natural raw material in the cosmetic products and dentistry and can be used in these fields.

When considering the limited scientific studies conducted on E. foetida, there is no study in the literature about its proliferative effects. Therefore, our study has shown for the first time that the Red Californian worm extract, which has been scientifically proven to be effective and which is a new and effective and sustainable natural raw material, can be used as a raw material due to its effects such as removing wrinkles (anti-wrinkle) and increasing the differentiation in the bone cells.

The present invention relates to the use of the Eisenia foetida extract in the cosmetic and regenerative fields. The Eisenia foetida extract is a single extract or a mixture of at least two extracts obtained from the solid or liquid fertilizers or from the worm itself.

The solid fertilizer, liquid fertilizer, and worm itself were used to obtain the extracts to be used in the present invention. The solid fertilizer and worm samples were kept in three different solvents (EtOH, ethanol), EA (ethyl acetate), DCM (dichloromethane) with different polarities for two days by mixing with the aid of a magnetic stirrer, after which the resulting extracts were filtered and evaporated in a rotary evaporator and freed from the solvents. The liquid fertilizer was lyophilized for use.

The Eisenia foetida extracts of the invention can be used as a wound healer, anti-inflammatory agent, and bone differentiation enhancer in the regenerative field. The effect on the mesenchymal stem cells obtained from the 3^rd molar dental pulp (DP-MSCs) derived from the neural crest was investigated and the DP-MSC viability showed a strong proliferative effect in 24 and 48 hours in the presence of the worm extracts. The extracts of the invention will provide the solutions especially for the dental diseases, the repair and treatment of the nerve injuries and the problems in the cosmetic and aesthetic field. In the results obtained, it has been determined that the extract increases the extracellular matrix production, improves the ossification, and preserves the cell sternness.

The Eisenia foetida extracts of the invention can be used in the cosmetic products, especially as an anti-aging agent. The extracts of different polarity were prepared in the laboratory from the solid and liquid fertilizers obtained from Eisenia foetida and/or the raw material obtained by drying the worm, and the inhibitory effect against the tyrosinase, elastase, collagenase, xanthine oxidase and cholinesterase enzymes was tested in vitro. Particularly, the inhibitory effect against collagenase supports the use of the worm extracts as an anti-aging agent in the cosmetic product formulation. Obtaining the Extracts

The extracts were prepared from the solid fertilizer, liquid fertilizer, and the worm itself obtained from Recep Tayyip ERDOGAN University, Rize. The solid fertilizer and worm samples were kept in three different solvents (EtOH, ethanol), EA (ethyl acetate), DCM (dichloromethane) with different polarities for two days by mixing with the aid of a magnetic stirrer. The extracts obtained at the end of this period were filtered and evaporated in a rotary evaporator and freed from the solvents. The liquid fertilizer was lyophilized and used in the studies. Determining the effect on the DP-MSCs (Dental Pulp Mesenchymal Stem Cells ) growth:

The different concentrations of the extracts (the liquid fertilizer and worm extract) were prepared in DMF10 (DMEM-LG, 10% fetal bovine serum, 1% Pen/Strep). The dental pulp mesenchymal stem cells (DP-MSCs) at 8000 cells/well were added to the 96-well microplates, the extracts of different concentrations were added following the cell adhesion and cultured for 24 and 48 hours. At the end of this period, the cell viability was determined colorimetrically using 2-(4-iodophenyl)-3-(4-nitrophenyl)-5-(2,4-disulphophenyl)-2H-tetrazolium (WST-1) (Abeam) following the manufacturer's instructions. The continuation studies were carried out for the worm extract that gave the best results.

The liquid fertilizer extract (GSH) and worm ethanol extract showed 85% viability at 100 ng/mL at the end of 24 hours, while the rate increased to 90% at the end of 48 hours (Figures 1 and 2). The liquid fertilizer extract showed the best result at 1000 ng/mL at the end of 24 hours (Figure 3). The worm ethanol extract stimulated the cell viability by 23% at 20 pg/mL at the end of 24 hours compared to the control group (Figure 4). The worm ethanol extract was used in the ossification studies. Determining the effect on the DP-MSC ossification characteristics:

The DP-MSC ossification was investigated for 21 days with the ossification medium containing 20 pg/mL of the worm ethanol extract selected according to the results of DP-MSC growth. The method of Pittenger et al. (1999) was used in the study. Briefly, the medium was removed from the cells showing 70-80% spread in the growth culture medium. The cells washed with the phosphate buffer (PBS, Sigma) were treated with the osteogenic differentiation medium. The differentiation medium comprises 10% FBS, 10 mM glycerophosphate, 0.2 mM ascorbic acid, 100 nM dexamethasone and 1% Pen/Strep in DMEM-LG. The culture medium was changed every 3-4 days over 21 days. 3 different experimental groups were formed, namely the group to which the extract was added for each time the culture medium was changed (OST-CONT), the group to which the extract was added in the medium changes corresponding to days 4 and 14 of the differentiation (OST-4, 14) and the group without the extract (OST). On day 21, Alizarin Red staining was carried out. The calcium concentration was determined by DICA 500, the early differentiation marker osteonectin (ON) and late ossification marker osteocalcin (OCN) were determined by ELISA method.

The effect of the E. foetida ethanol extract on the DP-MSC ossification was followed by the Alizarin red staining method performed on days 4, 7, 14 and 21 (Olympos CKX41). It was determined that the red-stained matrix and the black-stained calcium granules were observed intensely in the group treated with E. foetida.

The effects of E. foetida on the DP-MSC ossification were quantitatively observed by following the calcium concentration with DICA 500 (Figure 5).

In the differentiation study which was followed by adding E. foetida to the ossification medium on days 4 and 14 (OST-4, 14) and for 21 days (OST-CONT), it was observed that the matrix staining was more intense in the OST-4, 14 group in the images of alizarin staining. It was observed that the calcium granules were more intense than the control and OST-CONT groups.

The calcium granules observed in the alizarin red staining were quantitatively confirmed by measuring the concentration (Figure 6). It was determined that the OST-4, 14 group has the highest calcium concentration.

The early ossification marker osteonectin (ON) and the late ossification marker osteocalcin (OCN) were investigated in the samples taken from the culture supernatants on day 21 by the Elisa Kit method. The osteonectin reaches the highest level in the culture supernatant on day 14 of the differentiation and decreases on day 21 due to its low stability. In Figure 7, the amount of ON was found to be the least in the OST 4, 14 group. This data shows that the differentiation begins early.

The osteocalcin begins to form from day 14 in the differentiation. It was detected at the highest level in the OST-4, 14 group. It shows that the cells have turned into the osteoblastic cells at the protein level by ELISA KIT. Determining the effect of the E. foetida extract in the extracellular matrix (ECM) components

The ECM Sircol collagen kit was used for the determination analysis of the cellular collagen amount in DP-MSCs stimulated in terms of ossification in a 12-well microplate. Briefly, it was incubated for 48 h at 48°C in 0.5 M acetic acid containing 0.1 mg/mL pepsin. The samples were added to the Sircol staining agent and formed the collagen staining complexes and precipitated from the unbound staining. After centrifugation, the pellet was washed once with the acid-salt washing agent and suspended with an alkaline agent. The absorbance was measured at 555 nm by transferring 200 pL of solution to a 96-well microplate. The amount of collagen was determined according to the standard. 3 different experimental groups were formed, namely the group to which the extract was added for each time the culture medium was changed for 21 days in the ossification process (OST-CONT), the group to which the extract was added during the medium changes corresponding to days 4 and 14 of the differentiation (OST-4, 14) and the group without the extract (OST).

The effect of E. foetida on the collagen amount in DP-MSC extracellular matrix is shown in Figure 9. OST-4, 14 group was determined as the experimental group with the highest amount of collagen.

Results:

When E. foetida is used intermittently (days 1 and 14), DP-MSCs stimulated the osteogenic differentiation and ossification.

The ossification consists of the stages of the proliferation increase, differentiation, and formation of calcium granules. It is understood that when E. foetida is used intermittently in this mechanism, it increases the differentiation by increasing proliferation, increasing the amount of collagen, and inducing the formation of calcium granules.

The results showed that a single extract of Eisenia foetida obtained from the solid and/or liquid fertilizers of Eisenia Foetida and from the worm itself or a mixture of at least two extracts stimulated the osteogenic differentiation and ossification of the dental pulp mesenchymal stem cells.

It has been found that the E. foetida extract increases the calcium concentration and osteocalcin concentration to induce the ossification of the dental pulp mesenchymal stem cells. Further, the E. foetida extract increases the collagen concentration in the extracellular matrix.

Since the inhibitory effect of the Eisenia foetida extracts against the tyrosinase, elastase, collagenase, xanthine oxidase, and cholinesterase enzymes has been determined, it can be used especially in the anti-aging products.

The results obtained regarding the collagenase inhibitory effect and increasing the bone differentiation in dental mesenchymal stem cells indicate that the worm extracts can be a new and effective natural raw material for use in both the cosmetic product formulation and dental health.

The cosmetic and/or regenerative compositions can be the semi-solid preparations in the form of creams, ointments, gels, or the liquid preparations in the form of solutions. The compositions can be obtained according to the methods known in the pharmaceutical technology and in the cosmetics industry.

Description of the Figures:

Figure 1 : The viability results of DP-MSC treated with the GSH and EtOH extract for 24 hours

Figure 2: The viability results of DP-MSC treated with the GSH and EtOH extract for 48 hours

Figure 3: The viability results of DP-MSC treated with the GSH and EtOH extract for 24 and 48 hours

Figure 4: The viability results of DP-MSC treated with the E. foetida ethanol extract for 24 and 48 hours

Figure 5: Monitoring the effect of the E. foetida ethanol extract on the calcium concentration in the DP-MSC ossification

Figure 6: The calcium concentration in DP-MSCs directed to the ossification with E. foetida. Figure 7 : The amounts of osteonectin in DP-MSCs directed to the ossification with E. foetida.

Figure 8: The amounts of osteocalcin in DP-MSCs directed to the ossification with E. foetida. Figure 9: The effect of E. foetida on the amount of collagen in DP-MSC extracellular matrix.

Note: in Figures 6-8 OST: control group, OST-4, 14: DP-MSCs to which E.foetida is added on days 4 and 14, OST-CONT: DP-MSCs to which E. foetida is added for 21 days.

Claims

1. The Eisenia foetida extract, wherein it stimulates the osteogenic differentiation and ossification of the dental pulp mesenchymal stem cells.

2. The Eisenia foetida extract according to claim 1, wherein it is obtained from the solid or liquid fertilizers or from the worm itself.

3. The Eisenia foetida extract according to claim 2, wherein it is obtained from the solid fertilizers or from the worm itself by extracting the ethanol and then evaporating the solvent.

4. The Eisenia foetida extract according to claim 3, wherein it is obtained from the worm itself by extracting the ethanol and then evaporating the solvent.

5. The Eisenia foetida extract according to claim 2, wherein it is obtained from the liquid fertilizers by the lyophilization.

6. The Eisenia foetida extract according to claim 1, wherein it is a single extract or a mixture of at least two extracts obtained from the solid or liquid fertilizers or from the worm itself.

7. The Eisenia foetida extract according to claim 1, wherein it increases the calcium concentration for the ossification of the dental pulp mesenchymal stem cells.

8. The Eisenia foetida extract according to claim 1, wherein it increases the osteocalcin concentration for the ossification of the dental pulp mesenchymal stem cells.

9. The Eisenia foetida extract according to claim 1, wherein it increases the collagen concentration in the extracellular matrix.

10. The use of the Eisenia foetida extract in the products that provide the ossification and regeneration of the cell sternness in dentistry.

11. The use of the Eisenia foetida extract in the anti-aging products.