A SYNERGISTIC FORMULATION AGAINST ACNE AND A PROCESS FOR THE PREPARATION THEREOF

Abstract

The present invention relates to a synergistic formulation comprising the essential oils of plants from western Himalayas, which have versatile medicinal properties. The present invention also relates to a process for development of synergistic value added anti-acnes products using the essential oils from these plants of western Himalayas. The developed synergistic formulation is useful in inhibiting different P. acnes strains thereby exhibiting antibacterial activity.

Claims

1. A synergistic formulation comprising the essential oils of: (a) Valeriana jatamansi; (b) Tagetes minuta; (c) Curcuma aromatica; (d) Artemisia maritime; and (e) Hedychium spicatum; wherein the ratio between (a):(b):(c):(d):(e) ranges from 1 to 20:1 to 20:1 to 5:1 to 5:1 to 20.

2. The formulation as claimed in claim 1, wherein said formulation comprises the essential oils of Valeriana jatamansi:Tagetes minuta:Artemisia maritime:Hedychium spicatum in the ratio 10:5:5:20

3. The formulation as claimed in claim 1, wherein said formulation comprises the essential oils of Valeriana jatamansi:Tagetes minuta:Curcuma aromatica:Hedychium spicatum in the ratio 20:10:5:10.

4. The formulation as claimed in claim 1, useful for treatment of Acne vulgaris.

5. The formulation as claimed in claim 1, wherein said formulation can be diluted in the range of 0.0001 to 100% prior to application.

6. A process for the preparation of the formulation as claimed in claim 1, said process comprising: [a] mixing essential oils of Valeriana jatamansi, Tagetes minuta, Curcuma aromatic, Artemisia maritime, and Hedychium spicatum to obtain a mixture; and [b] homogenizing the mixture obtained in step [a] at temperature in the range of 15 to 25 ° C. for 1 to 3 hours to obtain the formulation; [c] optionally, diluting the formulation as obtained in step [b] by adding a diluting agent upto 10000 times prior to use.

7. The process as claimed in claim 6, wherein the essential oils are obtained from the aerial parts or from rhizomes or combination of both of aromatic crops selected from Valeriana jatamansi, Tagetes minuta, Curcuma aromatica, Artemisia maritime, and Hedychium spicatum.

8. The process as claimed in claim 6, wherein the essential oils are extracted using the method selected from the group consisting of solvent distillation, steam distillation, hydro distillation and hydro-steam distillation for a period in the range of 5 minutes to 96 hours.

9. The process as claimed in claim 6, wherein the diluting agent is selected from the group consisting of a non-polar solvent, a medium polar solvent, an organic solvent, an inorganic solvent, an emulsion, a commercial cream, a gel, and a lotion or combination thereof.

Description

DETAILED DESCRIPTION OF THE INVENTION

[0064] The present invention provides a synergistic formulation comprising essential oils of various plants from the Western Himalayas useful against acne. and the present invention also provides a process for the development of anti-acne products by using such synergistic formulation. The essential oils are obtained from the plants Tagetes minuta, Hedychium spicatum, Curcuma aromatic, Valeriana jatamansi, Dracocephalum heterophyllum and Artemisia maritime.

[0065] The process involved in the development of the claimed synergistic formulation comprises selection of different quality standardized essential oils from plants of western Himalaya and mixing them in different proportions with the help of a magnetic stirrer or by shaking manually or by mechanical shaker for two to three hours at room temperature. Then, the prepared synergistic formulation is optionally diluted with diluting agent selected from the group consisting of a non-polar solvent, a medium polar solvent, an organic solvent, an inorganic solvent, an emulsion, a commercial cream, a gel, a lotion or combination thereof along with different preservatives but not limited to binder, diluents, surfactants, perfumes and preservatives as a carrier or otherwise. The prepared mixture is further homogenized by using different devices suitable for homogenization.

[0066] The disclosed synergistic formulation is effectively useful for skin related problems, particularly for acne control. The formulation can be used for protection from pimples, blackheads, whiteheads, nodules, pustules, white heads and other skin related problems. The used essential oils and the synergistic formulation of the present invention were screened to characterize their therapeutic efficacy against the selected human P. acnes strains.

[0067] The essential oil of T. minuta is standardized against dihydrotagetone by Gas Liquid Chromatography (GLC). Similarly oils of H. spicatum, A. maritima, V. jatamansi, D. heterophyllum and C. aromatica were standardized against their phyto-chemical marker 1,8-cineol, camphor, patchouli alcohol, citronellol and borneol, respectively. All the oils were standardized for their biological activity against P. acnes strains isolated from skin acne lesions of Indian population. The present invention involves the selection and extraction of the essential oils by subjecting the parts of the plant to solvent extraction/hydro distillation/steam distillation or other method to isolate their active ingredients/extracts. The bioassay guided fractionation of the extracts was done to develop a synergistic formulation comprising essential oils of T. minuta, H. spicatum, C. aromatica, A. maritime, D. heterophyllum and V. jatamansi to yield a safe cosmeceutical composition for the treatment and control of acne and skin related problems, especially in human beings.

[0068] Bacterial Strains and Culturing Conditions

[0069] In this study, pathogenic microorganisms, Propionibacterium acnes HKG 300 (KY674888), Propionibacterium acnes HKG 304 (KY674892), Propionibacterium acnes HKG 277 (KY674865), Propionibacterium acnes HKG 315 (KY674903), Propionibacterium acnes HKG 306 (KY674894), Propionibacterium acnes HKG 309 (KY674897), Propionibacterium acnes HKG 314 (KY674902) were isolated at CSIR—IGIB [Institute of Genomics and Integrative Biology], Mathura Road, New Delhi from human skin acne lesions and used in the examples.

[0070] Propionibacterium acnes strains were individually inoculated in 1.0 ml of Brain Heart Infusion (BHI) broth (Himedia Laboratories Pvt. Ltd, India) followed by incubation in an anaerobic chamber to maintain the anaerobic environment under an atmosphere of 80% N.sub.2, 10% CO.sub.2, and 10% H.sub.2 at 37° C. The grown culture was further diluted in BHI broth to prepare a bacterial stock solution of concentration ˜10.sup.5 cfu/ml.

[0071] Minimum Inhibitory Concentration (MIC)

[0072] The Micro-broth dilution method was used in 96-well microtitre plates (Corning Technologies India Pvt. Ltd.) to obtain the MIC values of different essential oils and the synergistic formulation against various Propionibacterium acnes strains (Kumar B, Mathur A, Pathak R, Sardana K, Gautam H K, Kumar P. 2016 Evaluation of antimicrobial efficacy of quaternized poly[bis(2-chloroethyl) ether-alt-1,3-bis [3-(dimethylamino)propyl]urea] against targeted pathogenic and multi-drug-resistant bacteria. J Bioact Compat Polym 31: 467-480). To carry out the experiment, stock solution of essential oils and the synergistic formulation were prepared and diluted in the range of 0.001-0.5 mg/ml. P. acnes cultures along with appropriate amounts of essential oils were added in Corning® 96-well plates. Essential oils solutions were tested against pathogenic bacteria from higher to lower concentrations. Subsequently, absorbance in each well was measured at 600 nm in Tecan™ 96-well plate reader and incubated at 37° C. in anaerobic condition. The minimal concentration, at which microbial survival was not noticed, was considered as minimum inhibitory concentrations (MIC). On the basis of optical density (OD.sub.600 nm), the survival percentage of microbes was determined. The relative survival percentage of each microbe was used to calculate the MIC, which was determined using the following formula:

Relative survival %=[OD @ 600 nm of treated cells/OD @ 600 nm of nontreated cells (control)]×100

[0073] Levofloxacin was used as a positive control and culture medium without P. acnes culture and 5% aqueous DMSO with P. acnes culture was used as a negative control. The experiment was performed in triplicates. Standard deviation was analysed using the Microsoft Office Excel 2007.

EXAMPLES

[0074] The following examples are given by way of illustration only and therefore should not be construed to limit the scope of the present invention in any manner.

Example 1

[0075] The pathogenic microorganisms, Propionibacterium acnes HKG 300 (KY674888), Propionibacterium acnes HKG 304 (KY674892), Propionibacterium acnes HKG 277 (KY674865), Propionibacterium acnes HKG 315 (KY674903), Propionibacterium acnes HKG 306 (KY674894), Propionibacterium acnes HKG 309 (KY674897), Propionibacterium acnes HKG 314 (KY674902) were isolated at CSIR—IGIB [Institute of Genomics and Integrative Biology], Mathura Road, New Delhi from human skin acne lesions and used , as the test organisms. The test compounds are essential oils extracted from V. jatamansi (VJ), A. maritima (AM), D. heterophyllum (DH), T. minuta (TM) and H. spicatum (HS), respectively. The essential oils were dissolved in DMSO (5 mg/ml) and further diluted in autoclaved Milli Q water to make the working stock solution. All experiments were performed in triplicate. The results are shown in Table 3.

TABLE-US-00003 TABLE 3 Minimum Inhibitory Concentration (MIC) (mg/ml) of essential oil of various plants Samples KY674888 KY674892 KY674865 KY674903 KY674894 KY674897 KY674902 VJ 0.25 ± 0.04 0.25 ± 0.04 0.25 ± 0.04 0.31 ± 0.062  0.2 ± 0.058 0.25 ± 0.04 0.25 ± 0.11 AM 0.35 ± 0.07 0.43 ± 0.047 0.43 ± 0.047 0.45 ± 0.04 0.35 ± 0.07 0.31 ± 0.062 0.36 ± 0.047 DH >0.5 ± 0.00* >0.5 ± 0.00* >0.5 ± 0.00* >0.5 ± 0.00*  0.4 ± 0.081  0.4 ± 0.081 0.43 ± 0.047 TM >0.5 ± 0.00* >0.5 ± 0.00* >0.5 ± 0.00* >0.5 ± 0.00* >0.5 ± 0.00* >0.5 ± 0.00* >0.5 ± 0.00* CA 0.35 ± 0.07 0.43 ± 0.047  0.2 ± 0.058 0.43 ± 0.047 0.25 ± 0.04 0.43 ± 0.047 0.43 ± 0.047 HS 0.30 ± 0.12  0.2 ± 0.058 0.19 ± 0.05 0.25 ± 0.04  0.2 ± 0.058  0.2 ± 0.00 0.21 ± 0.023 *0.5 mg/ml was the highest concentration tested.

Example 2

[0076] This example used isolated Propionibacterium acnes strains KY674888, KY674892, KY674865, KY674903, KY674894, KY674897, and KY674902 from skin acne lesions as the test organisms. The details of P. acnes and essential oils are as described above. The test synergistic formulation 55A, 55D and 55E were obtained by mixing the essential oils from VJ, AM, TM, CA and HS at different ratios: wherein

[0077] The formulation 55A comprises the essential oils of VJ:TM:CA:AM:HS in the ratio of 1:1:1:1:1;

[0078] The formulation 55D comprises the essential oils of VJ:TM:CA:AM:HS in the ratio of 10:20:0:0:5 and

[0079] The formulation 55E comprises the essential oils of VJ:TM:CA:AM:HS in the ratio of 0:1:1:1:1.

[0080] The synergistic formulations were dissolved in DMSO (5 mg/ml) and further diluted in autoclaved Milli Q water to make the working stock solutions. All experiments were performed in triplicate. The results are shown in Table 4.

TABLE-US-00004 TABLE 4 Minimum Inhibitory Concentration (MIC) (mg/ml) of synergistic formulations prepared in Example 2 Samples KY674888 KY674892 KY674865 KY674903 KY674894 KY674897 KY674902 55A  0.2 ± 0.058 0.30 ± 0.081 0.125 ± 0.056 0.25 ± 0.04 0.21 ± 0.023 0.23 ± 0.047 0.4 ± 0.00 55D 0.26 ± 0.023 0.26 ± 0.023 0.083 ± 0.02  0.4 ± 0.08 0.21 ± 0.023 0.26 ± 0.023 0.4 ± 0.00 55E 0.25 ± 0.04 0.30 ± 0.07 0.166 ± 0.05 0.36 ± 0.047 0.25 ± 0.11 0.43 ± 0.047 0.4 ± 0.00

Example 3

[0081] This example used isolated Propionibacterium acnes strains KY674888, KY674892, KY674865, KY674903, KY674894, KY674897, and KY674902 from skin acne lesions as the test organisms. The details of P. acnes and essential oils are as described above. The test synergistic formulation 55B and 55C were obtained by mixing VJ, AM, TM, CA and HS essential oils at different ratios, wherein

[0082] The formulation 55B comprises the essential oils of VJ:TM:CA:AM:HS in the ratio of 10:5:0:5:20 and

[0083] The formulation 55C comprises the essential oils of VJ:TM:CA:AM:HS in the ratio of 20:10:5:0:10.

[0084] The synergistic formulation were dissolved in DMSO (5 mg/ml) and further diluted in autoclaved Milli Q water to make the working stock solution. All experiments were performed in triplicate. The results are shown in Table 5.

TABLE-US-00005 TABLE 5 Minimum Inhibitory Concentration (MIC) (mg/ml) of synergistic formulations prepared in Example 3 Samples KY674888 KY674892 KY674865 KY674903 KY674894 KY674897 KY674902 55B 0.26 ± 0.023 0.21 ± 0.023 0.125 ± 0.056  0.2 ± 0.058  0.2 ± 0.058 0.25 ± 0.00 0.25 ± 0.04 55C 0.25 ± 0.11 0.21 ± 0.023 0.125 ± 0.08 0.26 ± 0.023 0.125 ± 0.08 0.19 ± 0.05 0.31 ± 0.06

[0085] From the results obtained for examples 2 and 3, it was observed that the Formulations 55B and 55C exhibited best results in terms of anti-bacterial activity.

Example 4

[0086] GC-MS Analyses and Identification

[0087] The essential oils of each plant were analyzed with the help of GC/MS analyses. GC/MS analyses were conducted using a Shimadzu QP 2010 using a DB-5 (J&W Scientific, Folsom, USA) capillary column (30 m×0.25 mm i.d.; 0.25 μm thickness). Column temperature, 60° C. (3 minutes) to 240° C. (5 minutes) at 3° C./min. “Injector temperature, 250° C.”; “Interface temperature, 250° C.”; acquisition mass range, 800-50 amu; Detector Gain, 0.90 KV. Helium was used as carrier gas, 69.3 kPa (39.2 cm/s). Peak identification was accomplished by comparison of their mass spectral fragmentation pattern with those of reported in the literature (Adams, 1995) or by comparing with NIST library search.

[0088] GC Analyses (Quantitative Analysis)

[0089] The analysis of each essential oil was carried out by GC on Shimadzu GC-2010 equipped with a DB-5 (J&W Scientific, Folsom, USA) fusedsilica capillary column (30 m×0.25 mm i.d.; 0.25 μm film thickness). Column temperature, 90° C. (2 minutes) to 220° C. (5 minutes) with programming at 7° C./min. “Injector temperature, 240° C.”; “detector temperature, 260° C.”; injection mode, split. Carrier gas was helium at column flow rate of 1.05 ml/min (100 kPa). Retention indices (RI) of the sample components and authentic compounds were determined on the basis of homologous n-alkane hydrocarbons under the same conditions. The quantitative composition was obtained by peak area normalization and the response factor for each component was considered to equal 1. The results obtained in provided in Table 6.

TABLE-US-00006 TABLE 6 Chemical composition of essential oil from each plant. Constituents VJ AM DH TM CA HS Camphor — — — — 25.2 — Ocimene — — — 28.5 — — Borneol — 6.2 — — 3.7 — Isoborneol — — — — 8.0 — Dihydrotagetone — — — 22.9 — — Patchouli 42.3 — — — — — alcohol Eucalyptol — 33.4 — — — 65.0 Citronellol — — 75.2 — — —

ADVANTAGES OF THE INVENTION

[0090] Anti-acnes activity obtained because of the synergy of the essential oils. [0091] Cost effective process for development of anti-acnes product from naturally occurring essential oils. [0092] The process provides anti-acnes product that can be produced easily by pharmaceutical industries. [0093] Synergistic formulation has better fragrance compared to the starting materials and the new value added product can be used as a cure of acne as well as in cosmetics, fine fragrances, shampoos, toilet soaps and other toiletries. [0094] The synergistic formulation was developed by utilizing essential oils of local plants growing in Himachal Pradesh and hence will increase the market demand of the oils. The invention will thus enhance the value of these essential oils. [0095] T. minuta, H. spicatum, V. jatamansi, A. maritime, D. heterophyllum and C. aromatica grow abundantly in Himachal Pradesh and thus the present invention would provide an excellent opportunity to generate employment for plant collectors and also an opportunity for local people to cultivate these plants as an alternate cash crop for fulfillment of the market demand.