A PLANT EXTRACT AND COMPOUNDS FOR USE IN WOUND HEALING

Abstract

A plant extract, derived from a Salvia spp, may include one of at least one tanshinone compound, or at least one tanshinone compounds including a CYP11B1 inhibitory amount of at least one of tanshinone I and dihydrotanshinone. The plant extract may be used for use in the treatment of a wound or Cushing's syndrome.

Claims

1. A plant extract, derived from a Salvia spp, comprising one of: at least one tanshinone compound; or at least one tanshinone compound, including a CYP11B1 inhibitory amount of at least one of tanshinone I and dihydrotanshinone, wherein the plant extract is for use in the treatment of a wound or Cushing's syndrome.

2. A plant extract as claimed in claim 1, wherein the wound is a chronic wound.

3. A plant extract as claimed in claim 2, wherein the chronic wound is associated with diabetes.

4. A plant extract as claimed in claim 2, wherein the chronic wound is a venous or arterial ulcer.

5. A plant extract as claimed in claim 2, wherein the chronic wound is associated with prolonged pressure.

6. A plant extract as claimed in claim 2, wherein the chronic wound is associated with radiation burns.

7. A plant extract as claimed in claim 1, wherein the plant extract is for use in the treatment of Cushing's syndrome.

8. A plant extract as claimed in claim 1, comprising cryptotanshinone, dihydrotanshinone, tanshinone I, and tanshinone IIA, wherein the tanshinone I and the tanshinone IIA comprise at least 15%, by weight, of the plant extract, and the cryptotanshinon comprises at least 4%, by weight, of the plant extract.

9. A pharmaceutical or cosmetic comprising one of: at least one of tanshinone I and dihydrotanshinone, or an extract of Salvia spp containing at least one of tanshinone I and dihydrotanshinone, wherein the at least one of tanshinone I and dihydrotanshinone is in an amount that will inhibit CYP11B1 by at least 64%.

10. A pharmaceutical or cosmetic as claimed in claim 9, further comprising at least one excipient.

11. A pharmaceutical or cosmetic as claimed in claim 9, further comprising a carrier material.

12. A pharmaceutical or cosmetic as claimed in claim 9, wherein the pharmaceutical or cosmetic is used for periodontal applications.

13. A method of treating a wound or Cushing's syndrome, the method comprising providing a patient with one of: a therapeutically effective amount of a Salvia spp plant extract, or at least one tanshinone compound including a CYP11B1 inhibitory amount of at least one of tanshinone I and dihydrotanshinone.

14. A method as claimed in claim 13, wherein the wound is a chronic wound.

15. A pharmaceutical or cosmetic as claimed in claim 9, wherein the at least one of tanshinone I and dihydrotanshinone is in an amount that will inhibit CYP11B1 by at least 81%.

16. A pharmaceutical or cosmetic as claimed in claim 15, wherein the at least one of tanshinone I and dihydrotanshinone is in an amount that will inhibit CYP11B1 by at least 94%.

17. A pharmaceutical or cosmetic as claimed in claim 11, wherein the carrier material is one of a dressing and a bandage.

18. A pharmaceutical or cosmetic as claimed in claim 10, wherein the pharmaceutical or cosmetic is used for periodontal applications.

19. A pharmaceutical or cosmetic as claimed in claim 18, wherein the periodontal applications include at least one of mouthwash and toothpaste.

20. A pharmaceutical or cosmetic as claimed in claim 12, wherein the periodontal applications include at least one of mouthwash and toothpaste.

Description

BRIEF DESCRIPTION OF THE DRAWINGS

[0071] FIG. 1 is a diagram illustrating the biosynthetic pathway of steroid hormones in humans; and

[0072] FIG. 2 is an HPLC chromatogram of an extract according to the invention.

DETAILED DESCRIPTION

[0073] Applicant has discovered that Salvia miltiorrhiza Bunge extract (as disclosed in WO2009050451) inhibits CYP11B1 activity in intact cells in a dose dependent manner.

[0074] The extract disclosed in WO2009050451 is a selectively purified tanshinone compounds containing extract from the root of a Salvia spp comprising: [0075] Cryptotanshinone, [0076] Dihydrotanshinone, [0077] Tanshinone I, and [0078] Tanshinone IIA,
characterized in that the above identified tanshinone compounds comprise at least 15%, by weight, of the selectively purified extract, and the cryptotanshinone comprises at least 4%, by weight, of the selectively purified extract.

[0079] However, whilst the Salvia spp of WO2009050451 is Salvia miltiorrhiza Bunge, other Salvia spp such as: Salvia apiana, Salvia argentea, Salvia arizonica, Salvia azurea, Salvia camosa, Salvia clevelandii, Salvia coccinea, Salvia divinorum, Salvia dorrii, Salvia farinacea, Salvia forreri, Salvia fulgens, Salvia funerea, Salvia glutinosa, Salvia greggii, Salvia guaranitica, Salvia hispanica, Salvia leucantha, Salvia leucophylla, Salvia libanotica, Salvia longistyla, Salvia lyrata, Salvia mexicana, Salvia officinalis, Salvia patens, Salvia polystachya, Salvia potus, Salvia pratensis, Salvia roemeriana, Salvia sclarea, Salvia spathacea, Salvia splendens, Salvia verticillata, Salvia vitidis may be used to obtain a tanshinone containing extract.

[0080] Thus, the extract disclosed in WO2009050451 comprises at least 35%, by weight, of the identified tanshinone compounds with cryptotanshinone comprising at least 15%, by weight, of the selectively purified extract.

[0081] Indeed, preferably the identified tanshinone compounds comprised at least 45%, by weight, of the selectively purified extract, and the cryptotanshinone comprised at least 25% by weight, of the selectively purified extract.

[0082] In one embodiment the cryptotanshinone comprised at least 20%, more preferably at least 25%, more preferably still at least 40% and maybe as much as 60% of the four identified tanshinone compounds.

[0083] Similarly, the tanshinone IIA preferably comprised less than 55% of the four identified tanshinone compounds, more preferably still less than 50%, yet more preferably still less than 40% and might comprise as little as 20% or less of the four identified tanshinone compounds.

[0084] Most preferably the extract contains at least 1%, more preferably still at least 2% and more preferably still at least 3% of more of tanshinone I and/or dihydrotanshinone. Indeed the extract may be a highly selective extract containing at least 5%, more preferably at least 10%, through 20%, 30%, 40%, 50%, 60%, 70%, 80% and 90% of the one or more preferred compounds tanshinone I and/ or dihydrotanshinone.

[0085] In the embodiment exemplified in Example 1, the selectively purified tanshinone compound containing extract was characterized in that it comprises the four identified tanshinone compounds in an amount of 42.89% (plus or minus 40%, through 30% to 20%): [0086] a cryptotanshinone content of 18.95% (plus or minus 40%, through 30% to 20%), [0087] a dihydrotanshinone content of 3.65% (plus or minus 40%, through 30% to 20%), [0088] a tanshinone I content of 3.82% (plus or minus 40%, through 30% to 20%), and [0089] a tanshinone IIA content of 16.47% (plus or minus 40%, through 30% to 20%).

[0090] This selectively purified tanshinone compound containing extract was characterized in that it has an HPLC fingerprint substantially as illustrated in FIG. 2 with characteristic peaks as indicated.

[0091] However, it will be apparent from Example 3 (herein) that, whilst the extract is a potent CYP111B1 inhibitor, two of the lesser present Tanshinones, 15,16-dihydrotanshinone and tanshinone I are significantly more active than the major tanshinones present, cryptotanshinone and tanshinone IIA, and consequently it may be preferred to use alternative extracts with higher contents of one or more of the 15,16-dihydrotanshinone or tanshinone I, or indeed use the isolated compounds (or synthetically manufactured compounds or derivatives) either alone or together with one another.

[0092] Similarly, whilst the extract described above was prepared from the root of a Salvia spp comprising the steps of: [0093] soaking raw material in strong ethanol, for a time sufficient to solublize the tanshinone compounds, [0094] extracting the tanshinone compounds containing fraction using a percolation method, and [0095] concentrating the desired fraction under vacuum, and recovering the ethanol
it may be preferable to modify the process to concentrate or preferentially select the 15,16-dihydrotanshinone or tanshinone I.

[0096] Thus, alternative methodology to that disclosed in WO2009050451, namely utilising a first purification step comprising: [0097] a. dissolving the extract in sufficient water, [0098] b. allowing the desired fraction to precipitate out, [0099] c. discarding the aqueous solution, and [0100] d. collecting the precipitate.
might be used.

[0101] Similarly, whilst WO2009050451 discloses a second purification comprising [0102] e. a separation on a macroporous resin column (AB 8 macroporous resin column, manufactured by Lioayuan New Materials Ltd, or another suitable column) alternative methodology with specificity to the preferred compounds may be desired.

[0103] Details of the experiments supporting the claims are set out below:

EXAMPLE 1

1.1. Preparation of the Extract Solutions

[0104] Applicant dissolved 10 mg of the Salvia m. Bunge extract (as disclosed in WO2009050451) in the required volume of 100% ethanol or 100% DMSO to obtain a 1% (w/v) extract solution. They tested 5 L of this solution in a 500 L assay incubation volume (final ethanol or DMSO conc. of 1%). From this 1% Salvia m. Bunge extract solution, they also prepared a 1:10 and 1:100 dilution in 100% ethanol or 100% DMSO. From these solutions, they tested 5 L in a 500 L assay incubation volume.

[0105] 1.2. CYP11B1 Assay

[0106] The V79MZh11B1 cell line, expressing recombinant human CYP11B1, was cultured in Dulbecco's modified Eagle (DME, Sigma) medium supplemented with 5% fetal calf serum (FCS; Sigma), penicillin G (100 U/ml), streptomycin (100 g/ml), glutamine (2 mM) and sodium pyruvate (1 mM) at 37 C. in 5% CO.sub.2 in air. Cells were placed on 24-well cell culture plates (810.sup.5 cells per well) and cultured in 1 ml DME medium per well until confluence. On the day of testing, DME medium was removed and 450 l of fresh DMEM, containing 5 l of the extract solution in 100% ethanol or 100% DMSO, was added to each well. There was no significant difference in CYP11B1 inhibition between DMSO or ethanol as solvent. Control wells (receiving vehicle or ketoconazole (final concentration of 50 nM) as reference compound to validate each experiment) were treated in the same way without extract solution. After 60 min at 37 C. in the CO.sub.2 incubator, the reaction was started by the addition of 50 l of DMEM containing 100 nM of 11-deoxycorticosterone (plus 0.15 Ci of [1,2-.sup.3H] 11-deoxycorticosterone) as substrate. All measurements were in duplicate. After 25 min, the enzyme reaction was stopped by extracting the supernatant with ethyl acetate. Samples were centrifuged (10,000g, 10 min), and the upper phase was pipetted into fresh cups. The ethylacetate solvent was evaporated and the residue was dissolved in 40 l of methanol and analyzed by HPLC. The following formulas were used to determine the level of conversion and percentage of enzyme inhibition.

[00001]$\mathrm{CYP}.Math..Math.11.Math..Math.B.Math..Math.1$$\mathrm{Conversion}=100.Math.\%*\frac{\mathrm{area}\left(\mathrm{corticosterone}\right)}{\mathrm{area}\left(11-\mathrm{deoxycorticosterone}+\mathrm{corticosterone}\right)}$$\%.Math..Math.\mathrm{Inhibition}=100.Math.\%-\frac{\mathrm{conversion\_with}.Math.\mathrm{\_inhibitor}}{\mathrm{conversion\_without}.Math.\mathrm{\_inhibitor}}$

Results

[0107] The results are presented in Table 1 which shows the inhibition of CYP11B1 activity by Salvia m. Bunge extract in V79MZh11B1 cells. The extract solutions were freshly made from dry extract at the day of the experiment.

TABLE-US-00001 TABLE 1 Table 1. Determination of CYP11B1 inhibition by Salvia m. Bunge extract. The extract solutions were freshly made from dry extract at the day of the experiment in either 100% ethanol or 100% DMSO. N denotes the number of independent experiments. % Final extract conc. CYP11B1 inhibition Number of in CYP11B1 assay (mean SD) experiments Ethanol 0.0001% 22.6 9.9% N = 5 0.001% 79.1% N = 1 0.01% 95.6 4.4% N = 6 DMSO 0.0001% 16.8 1.5% N = 2 0.001% 83.1 11.4% N = 2 0.01% 100.0% N = 1

[0108] As shown, the Salvia m. Bunge extract prepared in 100% ethanol and 100% DMSO at a final concentration of 0.01% inhibited human CYP11B1 by 95.6% and 100.0%, respectively. From the 1% extract solutions, Applicant made a 1:10 dilution in 100% ethanol or 100% DMSO, respectively. From these latter solutions, they tested 5 L in 500 L assay volume. These extract solution (final extract concentration of 0.001% in the assay) inhibited human CYP11B1 by 79.1% and 83.1%, respectively. From the 1% extract solutions, Applicant made also 1:100 dilution in 100% ethanol or 100% DMSO, respectively. From the latter solution, they tested 5 L in 500 L assay volume. This extract solution (final extract concentration of 0.0001% in the assay) inhibited human CYP11B1 by 22.6% and 16.8%, respectively.

[0109] The conclusion from these experiments was that Salvia m. Bunge extract inhibits CYP11B1 at a dilution of 0.0001%, 0.001% and 0.01%.

[0110] In order to check that the inhibition was not due to toxicity, a MTT [3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl-tetrazolium bromide] cellular viability assay was performed in the same cell line under the incubation conditions used in the CYP11B1 screening assay as set out in Example 2 below:

EXAMPLE 2

MTT Cellular Viability Assay

[0111] V79MZh11B1 cells were cultured on 24-well cell culture plates (810.sup.5 cells per well) in 1 ml DME medium until confluence. On the day of testing, DME medium was removed and 450 l of fresh DME medium with 5% FCS, containing 5 l of the Salvia m. Bunge extract solution in 100% ethanol, was added to each well. Ethanol (1%) and Triton X-100 (0.0006%) were used as vehicle and positive control (all final concentrations), respectively. All measurements were in quadruplicate. After 60 min at 37 C. in a 5% CO.sub.2, 50 l of fresh DME medium (+5% FCS) was added to each well. After 25 min, medium was replaced by 500 l fresh DME medium (+5% FCS) to which 25 l of MTT solution (5 mg per ml PBS, pH 7.2) was added immediately. After 30 min, all medium was removed and the cells were lysed in 250 l of 0.5% acetic acid (v/v), 10% SDS (w/v) in DMSO. Absorbance of formazan was measured spectrophotometrically at 570 nm wavelength

[0112] Results

[0113] Determination of the Effect of 0.01% and 0.0001% Salvia m. Bunge Extract Solution on Cellular Viability of V79MZh11B1 Cells

[0114] The effect of Salvia m. Bunge extract on cellular viability of V79MZh11B1 cells under the (pre-) incubation conditions used in the CYP11B1 screening assay was determined. As shown in Table 2, the 0.01% and 0.0001% Salvia m. Bunge extract solutions had no effect on the conversion of MTT into formazan (whereas the positive control, Triton X-100, did almost fully block formazan formation). Therefore, it was concluded that the inhibitory effect of Salvia m. Bunge extract on CYP11B1 is not caused by a cytotoxic effect.

TABLE-US-00002 TABLE 2 Table 2. Lack of effect of Salvia m. Bunge extract on viability of V79MZh11B1 cells in the MTT toxicity assay. The extract solutions were freshly made from dry extract at the day of each experiment. MTT conversion into formazan, in the presence of 1% ethanol only, was set at 100% (data are mean SD). Final conc. Experiment nr. Treatment in assay Cell viability Experiment 1 Salvia m. bunge extract 0.01% 111 2% Salvia m. bunge extract 0.0001% 101 1% Ethanol (vehicle) 1% 100 3% Triton X-100 (pos. control) 0.0006% 3 1% Experiment 2 Salvia m. bunge extract 0.01% 124 7% Salvia m. bunge extract 0.0001% 122 11% Ethanol (vehicle) 1% 100 14% Triton X-100 (pos. control) 0.0006% 3 1%

EXAMPLE 3

[0115] Given the activity of the extract the Applicant looked at the activity of some of the tanshinones using the methodology described in Example 1.

[0116] The tanshinones tested in V79MZh11B1 cells were: [0117] tanshinone IIA, [0118] tanshinone I, [0119] dihydrotanshione I, and [0120] cryptotanshinone.

[0121] The results are shown in Table 3 below:

TABLE-US-00003 TABLE 3 Table 3. CYP11B1 inhibitory effect of tanshinone IIA, tanshinone I, dihydrotanshione I and cryptotanshinone. The results are mean SD of 2 independent experiments. Tanshinone IIA % CYP11B1 Tanshinone I % CYP11B1 Concentration Inhibition Concentration Inhibition 1 M 5.9 0.2 1 M 19.1 7.9 10 M 16.1 13.5 10 M 63.7 4.5 100 M 29.4 0.4 100 M 81.1 6.9 Dihydrotanshinone % CYP11B1 Cryptotanshinone % CYP11B1 Concentration Inhibition Concentration Inhibition 1 M 43.3 14.7 1 M 6.4 1.6 10 M 93.6 9.1 10 M 15.4 10.3 100 M 100.0 0.0 100 M 59.9 22.0

[0122] It will be apparent from the results that each of the tanshinones exhibited inhibitory activity, with the two most effective ones being dihydrotanshinone (94% inhibition at 10 M) and Tanshinone I (64%-inhibition at 10 M).

[0123] This in itself was unexpected, since these two compounds are present in lower amounts in the extract disclosed in WO2009050451 (respectively 3.65% and 3.82%) than cryptotanshinone and Tanshinone Ila (18.95% and 16.47% respectively).

[0124] Looking at the structures, it is possible that the enhanced activity of dihydrotanshinone (94% inhibition at 10 M) and Tanshinone I (64% inhibition at 10 M) might be attributed to the presence of a methyl (as opposed to a dimethyl) grouping at the C4 position.

[0125] Given the activity of these structurally related compounds, it is likely that other members of the Tanshinone family of compounds (or derivatives thereof) might be expected to exhibit similar (or better) CYP11B1 inhibitory activity.

EXAMPLE 4

[0126] Test for Thermal Stability at 70 C., 80 C. and 90 C. of Salvia m. Bunge Extract

[0127] In general, wound plaster constituents are frequently briefly held at elevated temperatures (70 C.-90 C.) to reduce the number of potential residual germs. It is therefore important that the CYP11B1 inhibitory activity of the extract/tanshinones should be stable at these elevated temperatures if they are to be used in situations where plaster is placed around a wound postoperatively.

[0128] The CYP11B1 inhibitory potency of the Salvia m. Bunge extract was determined after 5 min and 15 min of treatment at 70 C., 80 C. or 90 C. Salvia m. Bunge extract was dissolved in a 100% DMSO solution (at a concentration of either 0.05% and 0.025%) and incubated at 70 C., 80 C. and 90 C. for either 5 or 15 min, followed by testing in the CYP11B1 assay at a final concentration of 0.0005% and 0.00025%. These concentrations were chosen around the IC50 of the extract which inhibits CYP11B1.

[0129] The results are illustrated in Table 4 below which shows the thermal stability of the Salvia m. Bunge extract. Data are the meanSD of either 4 (control) or 2 measurements:

TABLE-US-00004 TABLE 4 Table 4. Thermal stability of the Salvia m. Bunge extract. Data are the mean SD of either 4 (control) or 2 measurements. Pretreatment of % Cyp11b1 inhibition 0.05% or 0.025% 0.0005% 0.00025% solution dilution dilution Control (25 C.) 42.2 2.5% 23.6 4.2% 70 C., 5 min 43.3 0.6% 25.2 3.8% 80 C., 5 min 43.2 1.8% 25.0 13.4% 90 C., 5 min 40.6 5.6% 27.3 3.4% 70 C., 15 min 45.8 0.6% 24.1 1.9% 80 C., 15 min 42.7 7.4% 30.2 2.6% 90 C., 15 min 44.4 3.6% 29.5 2.4%

[0130] No effect on CYP11B1 inhibitory activity was seen in any pretreatment at 70 C., 80 C. and 90 C. in comparison to control values (which were determined with the Salvia m. Bunge extract pretreated at 25 C. at a concentration of 0.05% and 0.025% for 15 min). Accordingly, these extracts/compounds may be used in situations where the wound is set in plaster.

EXAMPLE 5

[0131] Test for Allergenicity of the Salvia m. Bunge Extract.

[0132] Potential allergenicity of the extract was tested on intact human skin (inside of the upper arm) in three volunteers at a Salvia m. Bunge concentration of 0.5% (weight/volume) in 100% Vaseline. No sign of allergenicity (i.e. change in skin colour or texture) was seen in any individual during the five days of skin exposure.

[0133] From the above Examples it can be concluded that a plant extract, derived from a Salvia spp, comprising one or more tanshinone compounds, or said one or more tanshinone compounds, look promising candidates for use in the treatment of wounds or other conditions benefiting from inhibition of cortisol synthesis.

[0134] In addition, Applicant has identified that the two particularly active constituents are rather lipophilic (the log calculated using ACD/log P GALAS is 3.57 for dihydrotanshinone I), suggesting a good penetration to the epidermis which is essential to an efficient inhibition of the epidermally expressed target enzymes.