Synergistic combination for acne and method for preparing the same

Abstract

The present invention relates to a synergistic combination comprising essential oils from plants of the Western Himalayas with diverse medicinal properties. The present invention also relates to a method for developing synergistic value-added anti-acne products using essential oils from these plants of the Western Himalayas. The developed synergistic combination is useful in inhibiting different P. acnes strains, thereby exhibiting antibacterial activity.

Description

Technical Field

[0001] The present invention relates to a synergistic composition containing essential oils and a method for preparing the same. The prepared composition is useful for the development of value-added anti-acne products. The present invention further relates to the development of a composition showing specific anti-acne activity using essential oils of plants from the Western Himalayan region such as Tagetes minuta, Hedychium spicatum, Curcuma aromatica, Valeriana jatamansi, Dracocephalum heterophyllum and Artemisia maritima. The synergistic composition of essential oils developed in the present invention finds utility in perfumes, fragrances, pharmaceuticals, skin care, food, pesticides and other household or agricultural applications. Description of the background and prior art of the invention

[0002] Acne or acne vulgaris is a common skin disease of the sebaceous gland unit that affects approximately 90% of the adolescent population in Western European countries (Barefoot S F, Ratnam P. 1999 Composition and method for treating acne. US 5981473A). In India, 38.13% of girls and 50.6% of boys aged 12 - 17 years suffer from this disease (Epidemiology of acne 2009 Indian J Dermatol Venereol Leprol.75). Acne, a common skin disorder, has been found to increase the incidence of suicidal thoughts in acne patients (7.1%) (Kumar B, Pathak R, Mary P B, Jha D, Sardana K, Gautam H K 2016 New insights into the etiology of acne: exploring the role of the acne-associated microbiota. Dematologica Sinica 34:67 - 73).

[0003] According to the American Academy of Dermatology, multiple forms of acne occur. The most common are the forms of acne such as blackheads, whiteheads, and pustules, and the severe acne conditions including bacterial infections of papules, nodules, and cysts (Woody S T. 2016 Methods for preventing and treating acne. US 2016 / 0184220A1). Because there are many sebaceous glands, the parts of the body mainly affected by acne are the face, chest, neck, and upper shoulders (Barefoot S F, Ratnam P. 1999 Compositions and methods for treating acne. US 5981473A). There are many factors involved in the development of acne lesions such as bacterial colonization of the pilosebaceous duct, sebum production, inflammation, and ductal keratinization (Jappe U T A. 2003 Pathological mechanisms of acne and related therapies with particular emphasis on Propionibacterium acnes. Acta Derm Venereol 83:241-248) The main inducer of this multifactorial disease is the microbial variation of common resident microorganisms on the skin, and each microorganism has its own purpose and style of protecting the human body (Kumar B, Pathak R, Mary P B, Jha D, Sardana K, Gautam H K. 2016 New insights into the etiology of acne: exploring the role of acne-related microbial populations. Dermatologica Sinica 34:67-73).

[0004] Propionibacterium acnes (P. acnes) is a Gram-positive anaerobic bacterium that plays a major role in the development of acne (McDowell A, Patrick S, Eishi Y, lambert P, Eady A. 2013 Propionibacterium acnes in human health and disease. BioMed Research International 2013:3p). The growth of P. acnes in the environment is caused by the mixing of excessive sebum and follicular cells, and generates chemotactic factors and pro-inflammatory mediators that may cause inflammation (Mitra S K, Saxena E, Babu U V. 2010 Herbal acne control composition, its manufacturing method and its use. US 7785638B2).

[0005] There are many different therapies, including topical retinoids, topical or synthetic antibiotics or hormone therapy, or oral isotretinoin, which are mainly available for acne and depend on the type of clinical lesions. Benzoyl peroxide or topical antibiotics such as erythromycin, clindamycin, oral isotretinoin, or combinations of all these mediators can be used for mild to moderate inflammatory acne. However, today, some of these therapies are causing harmful side effects (Rathi S K. 2011 Treatment of acne vulgaris: current scenario. Indian J Dermatol 56:7-13).

[0006] Antibiotics such as clindamycin, minocycline, tetracycline, doxycycline, and erythromycin are frequently used by acne patients. However, the administration of these drugs causes many side effects such as abdominal cramps, black tongue, nausea, depression, fatigue, and dry skin (Barefoot S F, Ratnam P. 1999 Compositions and methods for treating acne. US 5981473A).

[0007] Worldwide, the high prevalence of antibiotic resistance is a problem for acne patients due to regional prescribing practices, patient compliance, and variability in the genome of P. acnes. In our laboratory's research, high resistance to antibiotics such as azithromycin (100%), erythromycin (98%), clindamycin (90.4%), doxycycline (444.2%), and tetracycline (30.8%) was revealed in the Indian population, but low resistance to minocycline (9.6%) and levofloxacin (9.6%) was observed. (Sardana K, Gupta T, Kumar B, Gautam H K, Garg V K. 2106 Cross-sectional pilot study of antibiotic resistance in P. acnes strains from acne patients in India using 16S rRNA polymerase chain reaction: comparison between therapies including antibiotics, benzoyl peroxide, and isotretinoin. Indian J Dermatol 61: 45-52) Antimicrobial resistance (AR) poses a serious threat to humanity worldwide. Treatments using the current array of therapies available in clinical practice have shown some cases of emerging AR. The discovery rate of antibiotics is very low compared to the increasing antibiotic resistance. The situation is worrisome and it is a strategic threat to the world. One approach to managing antibiotic resistance is to harness traditional knowledge and bring the potential of antibiotics to discovery platforms.

[0008] Essential oils are well-known for their versatile pharmaceutical properties. An essential oil is a concentrated hydrophobic liquid containing volatile aromatic compounds from plants. Essential oils are also known as volatile oils, ethereal oils, ethereal oleoresins, or simply the "oils" of the plants from which they are extracted. Various essential oils have been used medicinally at different times in history. The proposed medical uses for medicinal oils range from skin treatment to cancer treatment and, in many cases, are based solely on historical accounts of the use of essential oils for these purposes. According to a literature review, essential oils contain very good antibacterial activity (French medical system). The emergence of resistant strains associated with the strong use of antibacterial agents has led to the use of essential oils against microorganisms in vitro and in vivo (Warnke PH, Becker ST, Podschun R et al. J Craniomaxillofac Surg 2009; 37:392-397; Groppo FC, Ramacciato JC, Simoes RP, Florio FM, Sartoratto A. Int. Dent J 2002; 52:433-437). They are frequently used in dermatology, aromatherapy, and cosmetology worldwide to treat various conditions such as acne, dandruff, head lice, recurrent oral herpes, and oropharyngeal candidiasis. So far, several studies have analyzed the effects of pure essential oils and preservatives (Lakhdar L, Hmamouchi M, Rida S, Ennibi O. Odontostomatol Trop 2012; 35:38-46.) on bacteria and yeast (Carvalhinho S, Costa AM, Coelho AC, Martins E, Sampaio A. Mycopathologia 2012; 174:69-76).Most studies have shown that the use of essential oils as mouthwashes can reduce not only Streptococcus mutans but also the total amount of oral bacteria (Karbach J, Ebenezer S, Warnke PH, Behrens E., Al-Nawas B. Clin Lab 2015; 61:61-68). The exact composition of essential oils depends on the plant species of origin. They contain alcohols, esters, ethers, ketones, and aldehydes. They are essentially hydrophobic. This property allows them to partition and permeabilize the lipids of bacterial cell membranes and mitochondria, ultimately disrupting the cell structure (Devi KP, Nisha SA, Sakthivel R, Pandian SK. J. Ethnopharmacology, 2010; 130:107-15).

[0009] Therefore, considering the drawbacks of the prior art reported so far, the present invention aims to address the problem of multi-drug resistance of P. acnes due to the overuse of antibiotics. Objectives of the Invention

[0010] The main objective of the present invention is to provide a synergistic formulation containing essential oils of plants from the Western Himalayas.

[0011] Another objective of the present invention is to provide a simple and convenient method for converting essential oils of plants from the Western Himalayas into anti-acne products.

[0012] Yet another objective of the present invention is to provide a method for preparing an anti-acne formulation from essential oils.

[0013] Still another objective of the present invention is to prepare various bioactive products from essential oils of plants from the Western Himalayas.

Summary of the Invention

[0014] Accordingly, the present invention provides a synergistic formulation comprising essential oils of plants from the Western Himalayas, mixed in different ratios.

[0015] In one embodiment of the present invention, a synergistic formulation is provided that contains essential oils of T. minuta, H. spicatulum, C. aromatica, A. maritima, D. heterophyllum, and V. jatamansi in different ratios ranging from 1 to 60% w / v.

[0016] In another embodiment of the present invention, a synergistic formulation is provided in which the active ingredients of the essential oils include 1,8-cineole, patchouli alcohol, osimen, dihydrotagetone, camphor, citronellol, isoborneol, and borneol.

[0017] In yet another embodiment of the present invention, (i) providing essential oils of plants from the Western Himalayas, (ii) mixing the essential oils of the plants provided in step (i) in different ratios to obtain a mixture, (iii) mixing the mixture obtained in step (ii) for 1 to 3 hours at room temperature within the range of 15°C to 25°C using a magnetic stirrer, (iv) diluting the mixture obtained in step (iii) by adding a diluent in a ratio of essential oil:diluent of 1:0 to 1:10,000 and mixing at room temperature for 1 to 2 hours, and (v) applying the mixture obtained in step (iv) to different P. acne strains to evaluate antibacterial activity, a method for preparing the formulation is provided.

[0018] In yet another embodiment of the present invention, the essential oil is obtained from the whole or ground parts of the aerial parts of T. minuta, A. maritima, D. heterophyllum and the rhizome parts of V. jatamansi, H. spicatulum, and C. aromatica.

[0019] In yet another embodiment of the present invention, the essential oils of T. minutum, A. maritima, H. spicatum, C. aromatica, D. heterophyllum and V. yatamanshi were extracted by steam distillation or hydrodistillation or a combination thereof for a time ranging from 5 minutes to 72 hours.

[0020] In yet another embodiment of the present invention, the diluent is selected from the group consisting of organic solvents and inorganic solvents or combinations thereof.

[0021] In yet another embodiment of the present invention, the organic solvent is selected from the group of hydrocarbons, alcohols, ethers, and esters.

[0022] In yet another embodiment of the present invention, the final formulation contains a preservative selected from the group consisting of benzoic acid, butylated hydroxyanisole (BHA), sodium benzoate, and parabens. If desired, a desiccant can also be added to the formulation.

[0023] In yet another embodiment of the present invention, the essential oil can be obtained from plants grown in other geographical regions.

[0024] In yet another embodiment of the present invention, the final formulation is active against different microorganisms.

[0025] In yet another embodiment of the present invention, the final formulation is in a concentrated form containing no solvent residues.

[0026] In yet another embodiment of the present invention, the final formulation is freely soluble in nonpolar or medium polar solvents and can also be mixed with emulsions, gels, lotions, or combinations thereof.

[0027] In yet another embodiment of the present invention, the formulation may also be useful in concentrated form or at various dilution rates in perfumes, fragrances, cosmetics, pharmaceuticals, foods, pesticides, and other household or agricultural applications, and is a synergistic formulation comprising the essential oils of T. minutata, A. maritima, H. spicatum, C. aromatica, D. heterophyllum, and V. jatamansi.

[0028] In yet another embodiment of the present invention, the commercial use of the formulation can be carried out for flavoring, fragrance, food, pharmaceutical, and related industries for aromatic, pharmaceutical, and pesticide products.

[0029] In yet another embodiment of the present invention, the development of skin care products is carried out by mixing the formulation in a non-polar solvent or a polar solvent, or by distribution by crystallization, fractionation, or distribution between a polar solvent and a non-polar solvent or derivatization for product development.

[0030] One embodiment of the present invention is (a) Valeriana jatamansi (b) Tagetes minutata (c) Curcuma aromatica (d) Artemisia maritima, and (e) Hedychium spicatum providing a synergistic formulation containing the essential oils of each of (a):(b):(c):(d):(e) in a ratio of 1 to 20:1 to 20:1 to 5:1 to 5:1 to 20.

[0031] In yet another embodiment of the present invention, a synergistic formulation is provided, the formulation containing the essential oils of Valeriana jatamansi:Tagetes minutata:Artemisia maritima:Hedychium spicatum in a ratio of 10:5:5:20.

[0032] In yet another embodiment of the present invention, a synergistic formulation is provided, the formulation comprising essential oils of Valeriana jatamansi: Tagetes minuta: Curcuma aromatica: Hedychium spicatum in a ratio of 20:10:5:10.

[0033] In yet another embodiment of the present invention, a synergistic formulation useful for the treatment of acne vulgaris is provided.

[0034] In yet another embodiment of the present invention, a synergistic formulation is provided, which can be diluted within the range of 0.0001 to 100% before application.

[0035] Yet another embodiment of the present invention provides a method for preparing the formulation, the method comprising (a) mixing essential oils of Valeriana jatamansi, Tagetes minuta, Curcuma aromatica, Artemisia maritima, Hedychium spicatum to obtain a mixture, and (b) homogenizing the mixture obtained in step (a) at a temperature within the range of 15 to 25 °C for 1 to 3 hours to obtain a formulation, (c) optionally diluting the formulation obtained in step (b) by adding a diluent up to 10,000 times before use.

[0036] Yet another embodiment of the present invention provides a method for preparing the formulation, wherein the essential oil is obtained from the aerial part or rhizome or a combination of both of a spice crop selected from Valeriana jatamansi, Tagetes minuta, Curcuma aromatica, Artemisia maritima, and Hedychium spicatum.

[0037] Yet another embodiment of the present invention provides a method for preparing the formulation, wherein the essential oil is extracted using a method selected from the group consisting of solvent distillation, steam distillation, hydrodistillation, and hydrosteam distillation for a time within the range of 5 minutes to 96 hours.

[0038] In yet another embodiment of the present invention, a method for preparing a formulation is provided, wherein the diluent is selected from the group consisting of nonpolar solvents, medium-polar solvents, organic solvents, inorganic solvents, emulsions, commercially available creams, gels, lotions, or combinations thereof.

[0039] In yet another embodiment of the present invention, a method for preparing a formulation is provided, wherein the nonpolar solvent is selected from the group consisting of short-chain, medium-chain, and long-chain hydrocarbons, fats, ethers, and other solvents having a dielectric constant of less than 15.

[0040] In yet another embodiment of the present invention, a method for preparing a formulation is provided, wherein the medium-polar solvent is selected from the group consisting of short-chain, medium-chain, and long-chain hydrocarbons, fats, ethers having one oxygen group, and other solvents having a dielectric constant of 15 to 50.

[0041] In yet another embodiment of the present invention, a method for preparing a formulation is provided, wherein the organic solvent is selected from the group consisting of hydrocarbons and other carbon-based chemicals.

[0042] In yet another embodiment of the present invention, a method for preparing a formulation is provided, wherein the inorganic solvent is selected from the group excluding carbon-based chemicals such as water, ammonia, and carbon dioxide.

[0043] In yet another embodiment of the present invention, a method for preparing a formulation is provided, wherein the emulsion is selected from the group consisting of mixtures of two or more immiscible liquids.

[0044] In yet another embodiment of the present invention, a method for preparing a formulation is provided, wherein the gel is selected from the group consisting of solids dispersed in a liquid medium and is capable of forming solids having a range of properties (from hard and tough to soft and weak).

[0045] In yet another embodiment of the present invention, a method for the preparation of a formulation is provided, wherein the lotion is selected from the group consisting of an aqueous liquid containing insoluble substances to an alcoholic liquid. Details of the biological resources used in the present invention

[0046] The biological resources used for the purposes of the present invention were plants collected from the CSIR-IHBT [CSIR-Himalayan Institute of Bioresource Technology] farm, Chandpura, Kangra district, Himachal Pradesh, India, either wild or. Complete details in this regard are given in the following tables: Tables 1 and 2.

[0047]

Table 1

[0048]

Table 2

[0049] 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 method for the development of anti-acne products by using such synergistic formulations. The essential oils are obtained from Plantago minuta, Hedychium spicatum, Curcuma aromatica, Valeriana jatamansi, Dracocephalum heterophyllum and Artemisia maritima.

[0050] The method involved in the development of the claimed synergistic formulation comprises selecting standardized essential oils of different qualities from plants from the Western Himalayas and mixing them in different ratios by shaking them at room temperature for 2 - 3 hours with the help of a magnetic stirrer or manually or with a mechanical shaker.

[0051] Next, the prepared synergistic formulation is optionally diluted with a diluent selected from the group consisting of non-polar solvents, medium-polar solvents, organic solvents, inorganic solvents, emulsions, commercially available creams, gels, lotions or combinations thereof, together with various preservatives, but is not limited to binders, diluents, surfactants, fragrances, and preservatives as carriers. The prepared mixture is further homogenized by using various devices suitable for homogenization.

[0052] The disclosed synergistic formulation is effectively useful for controlling skin-related problems, especially acne. This formulation can be used for protection from comedones, blackheads, whiteheads, nodules, pustules, milia and other skin-related problems. The essential oils used and the synergistic formulation of the present invention were screened to characterize their therapeutic effects against selected human P. acnes strains.

[0053] The essential oil of T. minutata is standardized against dihydrotagetone by gas-liquid chromatography (GLC). Similarly, the oils of H. spicatum, A. maritima, V. jatamansi, D. heterophyllum and C. aromatica are standardized against the phytochemical markers 1,8-cineole, camphor, patchouli alcohol, citronellol, and borneol, respectively. All oils were standardized for their biological activity against P. acnes strains isolated from acne lesions of the skin of Indian inhabitants. The present invention includes the selection and extraction of essential oils by subjecting plant parts to solvent extraction / hydrodistillation / steam distillation or other methods to isolate their active ingredients / extracts. Bioassay-guided fractionation of the extracts was performed to develop a synergistic formulation containing the essential oils of T. minutata, H. spicatum, C. aromatica, A. maritima, D. heterophyllum, V. jatamansi, especially to produce a safe pharmaceutical cosmetic composition for the treatment and control of human acne and skin-related problems.

[0054] Strains and culture conditions In this study, the pathogenic microorganisms Propionibacterium acnes HKG300 (KY674888), Propionibacterium acnes HKG304 (KY674892), Propionibacterium acnes HKG277 (KY674865), Propionibacterium acnes HKG315 (KY674903), Propionibacterium acnes HKG306 (KY674894), Propionibacterium acnes HKG309 (KY674897), and Propionibacterium acnes HKG314 (KY674902) were isolated from human skin acne lesions at CSIR-IGIB [Institute of Genomics and Integrative Biology] on Mathura Road, New Delhi.

[0055] Each Propionibacterium acnes strain was individually inoculated into 1.0 ml of Brain Heart Infusion (BHI) broth (Himedia Laboratories Pvt. Ltd, India), and then incubated in an anaerobic chamber at 37°C under an atmosphere of 80% N 2 , 10% CO 2 and 10% H 2 to maintain an anaerobic environment. The grown cultures were further diluted with BHI broth to prepare a bacterial stock solution with a concentration of 10 5 cfu / ml.

[0056] Minimum inhibitory concentration (MIC) The microbroth dilution method was used in a 96-well microtiter plate (Corning Technologies India Pvt. Ltd) to obtain the minimum inhibitory concentrations (MICs) of various essential oils and synergistic formulations against various Propionibacterium acnes strains. (Kumar B, Mathur A, Pathak R, Sardana K, Gautam HK, Kumar P. 2016 Evaluation of the antibacterial effect 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 conduct the experiments, stock solutions of essential oils and synergistic formulations were prepared and diluted within the range of 0.001 - 0.5 mg / ml. Appropriate amounts of P. acnes cultures were added to Corning® 96-well plates along with the essential oils. The essential oil solutions were tested from high to low concentrations against the pathogenic bacteria. Subsequently, the absorbance of each well was measured at 600 nm using a Tecan® 96-well plate reader and incubated at 37°C under anaerobic conditions. The minimum concentration at which no microbial survival was observed was considered the minimum inhibitory concentration (MIC). The survival rate of the microorganisms was determined based on the optical density (OD 600 ). The MIC was calculated using the relative survival rate of each microorganism. This was determined using the following formula.

[0057] Relative survival rate % = [OD@600nm of treated cells / OD@600nm of untreated cells (control)] × 100 Levofloxacin was used as a positive control and a medium without P. acnes culture, and 5% aqueous DMSO containing P. acnes culture was used as a negative control. The experiments were conducted three times. The standard deviation was analyzed using Microsoft Office Excel 2007. For example

[0058] The following examples are given by way of illustration only and should not be construed as limiting the scope of the invention in any way.

[0059] Example 1 The pathogenic microorganisms Propionibacterium acnes HKG300 (KY674888), Propionibacterium acnes HKG304 (KY674892), Propionibacterium acnes HKG277 (KY674865), Propionibacterium acnes HKG315 (KY674903), Propionibacterium acnes HKG306 (KY674894), Propionibacterium acnes HKG309 (KY674897), and Propionibacterium acnes HKG314 (KY674902) were isolated from human skin acne lesions at CSIR-IGIB [Genomics and Integrative Biology Institute] on Mathura Road, New Delhi, and used as test organisms. The test compounds were essential oils extracted from V. yatamanci (VJ), A. maritima (AM), D. heterophyllum (DH), T. minutum (TM), and H. spicatulum (HS), respectively. Each essential oil was dissolved in DMSO (5 mg / mL) and further diluted with autoclaved Milli Q water to prepare a working stock solution. All experiments were performed three times. The results are shown in Table 3. Table 3. Minimum inhibitory concentration (MIC) (mg / ml) of essential oils from each plant

[0060]

Table 3

[0061] Example 2 In this example, Propionibacterium acnes strains KY674888, KY674892, KY674865, KY674903, KY674894, KY674897, and KY674902 isolated from skin acne lesions were used as test organisms. The details of P. acnes and the essential oils are as described above. The test synergistic formulations 55A, 55D, and 55E were obtained by mixing essential oils from VJ, AM, TM, CA, and HS in different ratios. Here, Complex 55A contains essential oils of VJ:TM:CA:AM:HS in a ratio of 1:1:1:1:1, Complex 55D contains essential oils of VJ:TM:CA:AM:HS in a ratio of 10:20:0:0:5, Complex 55E contains essential oils of VJ:TM:CA:AM:HS in a ratio of 0:1:1:1:1:1.

[0062] Each synergistic compound was dissolved in DMSO (5 mg / ml) and further diluted with autoclaved Milli Q water to prepare a working stock solution. All experiments were performed three times. The results are shown in Table 4. Table 4. Minimum inhibitory concentration (MIC) (mg / ml) of the synergistic compounds prepared in Example 2

[0063] [Table 4]

[0064] Example 3 In this example, Propionibacterium acnes strains KY674888, KY674892, KY674865, KY674903, KY674894, KY674897, and KY674902 isolated from skin acne lesions were used as test organisms. The details of P. acnes and essential oils are as described above. The test synergistic compounds 55B and 55C were obtained by mixing each essential oil of VJ, AM, TM, CA, and HS in different ratios. Here, Complex 55B contains each essential oil of VJ:TM:CA:AM:HS in a ratio of 10:5:0:5:20, Complex 55C contains each essential oil of VJ:TM:CA:AM:HS in a ratio of 20:10:5:0:10.

[0065] The synergistic compound was dissolved in DMSO (5 mg / ml) and further diluted with autoclaved Milli Q water to prepare a working stock solution. All experiments were performed three times. The results are shown in Table 5. Table 5. Minimum inhibitory concentration (MIC) (mg / ml) of the synergistic combination adjusted in Example 3

[0066] [Table 5]

[0067] Example 4 Identification by GC-MS analysis The essential oils of each plant were analyzed using GC / MS analysis. The GC-MS analysis was carried out using a Shimadzu QP2010 with a DB-5 (J&W Scientific in Folsom, USA), capillary column (inner diameter 30 m × 0.25 mm, thickness 0.25 μm). The column temperature was from 60 °C (3 min) to 240 °C (5 min) at 3 °C / min. "Injector temperature 250 °C"; "Interface temperature 250 °C", acquisition mass range 800 - 50 amu, and helium at 69.3 kPa (39.2 cm / s) as the carrier gas with a detector gain of 0.90 KV was used. Peak identification was accomplished by comparing their mass spectral fragmentation patterns with those reported in the literature (Adams, 1995) or by comparison with NIST library search. GC analysis (quantitative analysis) The analysis of each essential oil was performed by GC of a Shimadzu GC-2010 equipped with a DB-5 (J&W Scientific in Folsom, USA), fused silica capillary column (inner diameter 30 m × 0.25 mm, film thickness 0.25 μm). The column temperature was programmed at 7 °C / min from 90 °C (2 min) to 220 °C (5 min). "Injector temperature 240 °C", "Detector temperature 260 °C", injection mode split. The carrier gas was helium with a column flow rate of 1.05 ml / min (100 kPa). The retention indices (RI) of the sample components and authentic compounds were determined based on homologous n-alkane hydrocarbons under the same conditions. The quantitative composition was obtained by normalization of the peak areas, and the response factor of each component was considered to be equal to 1. The results obtained are shown in Table 6. Table 6. Chemical composition of the essential oils from each plant

[0068]

Table 6

[0069] · Anti - acne activity obtained by the synergistic action of essential oils · A cost - effective process for developing anti - acne products from natural essential oils · This process provides anti - acne products that can be easily manufactured in the pharmaceutical industry. · The synergistic formulation has an excellent fragrance compared to the starting materials, and this new value - added product can be used not only for the treatment of acne but also in cosmetics, high - end fragrances, shampoos, cosmetic soaps, and other toiletries. · The synergistic formulation is developed using essential oils of local plants grown in the Himalayan Pradesh region, so the market demand for these oils will increase. Therefore, the present invention will enhance the value of these essential oils. · T. minuta, H. spicatum, V. jatamansi, A. maritima, D. heterophyllum, and C. aromaticum grow abundantly in the Himalayan Pradesh region. Therefore, the present invention provides an excellent opportunity to generate employment for plant collectors and an opportunity for local people to cultivate these plants as alternative cash crops to meet the market demand. The invention described in the claims of the present application at the time of original filing is appended below. [1] (a) Valeriana jatamansi (b) Tagetes minuta (c) Curcuma aromatica (d) Artemisia maritima, and (e) Hedychium spicatum A synergistic formulation containing each essential oil of (a):(b):(c):(d):(e) in a ratio ranging from 1 to 20:1 to 20:1 to 5:1 to 5:1 to 20. [2] The formulation according to [1], wherein the formulation contains each essential oil of Valeriana jatamansi:Tagetes minuta:Artemisia maritima:Hedychium spicatum in a ratio of 10:5:5:20. [3] The formulation according to [1], wherein the formulation contains each essential oil of Valeriana jatamansi:Tagetes minuta:Curcuma aromatica:Hedychium spicatum in a ratio of 20:10:5:10. [4] The formulation according to [1] useful for the treatment of acne vulgaris. [5] The formulation according to [1], wherein the formulation can be diluted within the range of 0.0001 to 100% before application. [6] [a] Mixing each essential oil of Valeriana jatamansi, Tagetes minuta, Curcuma aromatica, Artemisia maritima, and Hedychium spicatum to obtain a mixture, and [b] Homogenizing the mixture obtained in step [a] at a temperature within the range of 15 to 25 °C for 1 to 3 hours to obtain a formulation, [c] Optionally, diluting the formulation obtained in step [b] by adding a diluent up to 10,000 times before use A method for preparing the formulation according to [1] comprising the above steps. [7] The method according to [6], wherein the essential oil is obtained from the aerial part or its rhizome or a combination of both of a spice crop selected from Valeriana jatamansi, Tagetes minuta, Curcuma aromatica, Artemisia maritima, and Hedychium spicatum. [8] The method according to [6], wherein the essential oil is extracted using a method selected from the group consisting of solution distillation, steam distillation, hydrodistillation, and hydrosteam distillation for a time within the range of 5 minutes to 96 hours. [9] The method according to [6], wherein the diluent is selected from the group consisting of non-polar solvents, medium-polar solvents, organic solvents, inorganic solvents, emulsions, commercially available creams, gels, and lotions, or combinations thereof.

Claims

1. (a)Valeriana jatamansi (b)Tagetes minuta (c)Curcuma aromatica (d)Artemisia maritima, and (e)Hedechium spicatum comprising each essential oil of, and the ratio of (a):(b):(c):(d):(e) is ranging from 1 to 20:1 to 20:1 to 5:1 to 5:1 to 20, a synergistic composition for use in the treatment of acne vulgaris.

2. The composition according to claim 1, wherein the composition comprises each essential oil of Valeriana jatamansi:Tagetes minuta:Artemisia maritima:Hedechium spicatum in a ratio of 10:5:5:

20.

3. The composition according to claim 1, wherein the composition comprises each essential oil of Valeriana jatamansi:Tagetes minuta:Curcuma aromatica:Hedechium spicatum in a ratio of 20:10:5:

10.

4. The composition according to claim 1, wherein the composition can be diluted within the range of 0.0001 to 100% before application.

5. [a]mixing each essential oil of Valeriana jatamansi, Tagetes minuta, Curcuma aromatica, Artemisia maritima, Hedechium spicatum to obtain a mixture, and [b]homogenizing the mixture obtained in step [a] at a temperature within the range of 15 to 25 °C for 1 to 3 hours to obtain a composition, [c]optionally, diluting the composition obtained in step [b] by adding a diluent up to 10,000 times before use A method for preparing the composition according to claim 1, comprising.

6. The method according to claim 5, wherein the essential oil is obtained from the aerial part or its rhizome or a combination of both of a spice crop selected from Valeriana jatamansi, Tagetes minuta, Curcuma aromatica, Artemisia maritima and Hedechium spicatum.

7. The method according to claim 5, wherein the essential oil is extracted using a method selected from the group consisting of solution distillation, steam distillation, hydrodistillation and hydrosteam distillation for a time within the range of 5 minutes to 96 hours.

8. The method according to claim 5, wherein the diluent is selected from the group consisting of a nonpolar solvent, a medium polar solvent, an organic solvent, an inorganic solvent, an emulsion, a commercially available cream, a gel and a lotion, or a combination thereof.

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