Azadirachta indica leaf extract

Abstract

Compared with the relative average proportion content of the azadirachtin and the azadirachtin in the azadirachtin leaves, the azadirachtin extract provided by the invention is rich in the azadirachtin and / or lacks the content of the azadirachtin. The method for producing such an extract of the invention comprises administering a supercritical CO2 extraction under conditions that facilitate selective enrichment of azadirachtin content in the extract and / or selective reduction of azadirachtin content.

Description

[0001] The present invention relates to an extract of the leaves of the neem tree (Azadirachta indica), which is rich in certain components of the leaves of the neem tree, namely rich in nimbolide. In addition, the present invention relates to a method for obtaining the extract of the present invention and cosmetic and pharmaceutical compositions comprising such an extract. The present invention also relates to the use of the neem leaf extract of the present invention in skin care and dermatology and in other medical fields, wherein the extract can be used to treat and alleviate diseases and their symptoms.

[0002] Background and Prior Art

[0003] For thousands of years, the neem tree (Azadirachta indica) has been considered a medicinal plant. It is often described and considered as a "village pharmacy", especially in rural areas of India, however, it is also widely used in other parts of the world. Plant parts, such as leaves, bark and seeds, and products obtained therefrom, are used in various forms of application and are also established as a reservoir of Ayurvedic medicines. Many of the uses of neem products are based on the traditional knowledge of the people in countries where the neem tree has been grown for centuries.

[0004] Various plant parts are considered to be effective for different purposes, but also partially for the same or at least overlapping purposes. For example, neem oil obtained from the seeds of the neem tree is known for its use as an insecticide but also for cosmetic uses and purposes and as a natural spermicide. On the other hand, oral ingestion of neem oil is not recommended. Neem leaves are widely used in the cosmetic industry, but are also used in medicinal applications.

[0005] In the Western Hemisphere, scientific research was initiated by the work of Professor Schmutterer (e.g., summarized in: Heinrich Schmutterer (Hrsg.): The neem tree Azadirachta indica (A. Juss.) and other meliaceous plants. Sources of unique natural products for integrated pest management, medicine, industry and other purposes. VCH Verlagsgesellschaft, Weinheim 1995, ISBN 3-527-30054-6), who studied the use of neem products as insecticides after discovering that the neem tree was the only plant that survived a particularly terrible locust plague. His research focused primarily on azadirachtin and other plant components that showed insecticidal effects.

[0006] The real scientific literature on ingredients, isolation and use or application forms of such ingredients exists only to a very limited extent and is not conclusive. The studies reflected in such literature often do not follow scientific standards and the results described therein are often not reproducible. Often, there is also a lack of reliable analysis for comparing the observed effects. For example, the origin of neem leaves is often stated as "from our university garden", and the actual content of the ingredients in the source material is not analyzed at all, or at least not analyzed in a reproducible manner.

[0007] In addition to its use in traditional medicine, neem and certain specific components that can be enriched or isolated from the plant have recently been studied for the treatment of various diseases, especially for use in cancer therapy. Studies have shown unexpected effects of the use of such substances, and it is postulated that the anti-cancer effects are caused by the modulation of multiple cellular processes when neem extracts or purified or isolated neem components are applied.

[0008] One of these components of interest is azadirachtin, as it has shown activity against a variety of different types of cancer in in vivo and in vitro studies. Azadirachtin has also been shown to be effective in inducing apoptosis while inhibiting metastasis and angiogenesis. Additional components of the neem tree have been shown to have anti-inflammatory effects and other medically relevant properties.

[0009] Prior art describes various methods for obtaining extracts including various neem ingredients. For example, US2020 / 0069636 A1 summarizes some related prior art documents and methods described therein, which include extraction methods using specific solvents and solvent combinations and also partially include precipitation and chromatographic fractionation separation methods. Related documents include WO2015 / 035199A1, CN 101972246 B, CN 103864876 A, US 5,370,873 A, JP 2009 / 274956A and WO 2007 / 137389 A1. However, these methods are considered to be unsuitable for the use of the relatively high temperature (which is considered to destroy or change the molecular structure of the temperature-sensitive component with therapeutic value) for their separation. In addition, the use of solvents is considered to be undesirable because these solvents may have adverse effects on human health and even they themselves may be carcinogenic.

[0010] The authors of US2020 / 0069636 A1 propose a method for preparing a CO2 extract of neem, wherein heat-sensitive plant components are obtained during the extraction process by applying supercritical CO2 extraction conditions at a temperature below 45°C (which is known not to negatively affect heat-sensitive components). However, the method applied requires many process steps, including repeated extractions: more specifically, in addition to supercritical CO2 extraction, a mixture of CO2 and ethanol is used for further extraction, and in order to remove the solvent, vacuum distillation is required in the method. Therefore, the method disclosed in this document requires considerable procedural effort and still cannot completely eliminate the use of solvents in order to achieve a satisfactory yield of the desired neem components.

[0011] In the context of investigating the medical use of azadirachtin for various diseases and medical conditions, the use of commercially available azadirachtin can also be envisaged. However, especially when it comes to diseases that, although not life-threatening, require symptom relief and treatment, the use of such commercially available azadirachtin is not a preferred option, as the cost is very high for even very small amounts of the substance. Moreover, in such cases, the synergistic effect provided by the presence of other neem components is lost.

[0012] For many applications and especially for certain medical applications, several neem components such as azadirachtin are considered undesirable because they may also have negative effects on humans when applied or ingested, especially when applied over longer periods of time. Furthermore, for dermatological purposes, the inclusion of such substances is undesirable.

[0013] Therefore, the object behind the present invention is, on one hand, to provide a neem extract containing elevated concentrations of azadirachtin compared to known extracts and to provide a simple and cost-effective process for producing such an extract while avoiding the inclusion of undesirable substances from the neem plant as well as from the extraction process. SUMMARY OF THE INVENTION

[0015] As a first aspect of the present invention, the object has been solved by providing an extract of neem (Azadirachta indica) leaves, which is enriched in azadirachtin content as compared to the relative average proportion of this component in neem leaves, the extract containing 4 to 25 weight % of azadirachtin.

[0016] In a second aspect of the present invention, there is provided a process for producing the neem leaf extract of the present invention, the process comprising applying supercritical CO2 extraction under conditions conducive to selectively enriching the azadirachtin content in the extract as compared to the average proportional content of this component in the neem leaves, wherein the supercritical CO2 extraction is carried out at a temperature of 50°C to 100°C.

[0017] In a third and fourth aspect of the present invention, cosmetic compositions and pharmaceutical compositions are provided, which comprise the extract of the present invention as defined above. In a fifth aspect of the present invention, the extract of the present invention and the cosmetic or pharmaceutical composition of the present invention are provided for use in dermatology, the treatment of cancer diseases and the treatment of inflammatory disorders. DETAILED DESCRIPTION OF THE INVENTION

[0019] In the context of their studies, the present inventors were able to establish a process that allows the extraction of azadirachtin and some additional constituents of neem leaves in a highly efficient and selective manner.

[0020] In particular, the azadirachtin content in the neem extract of the present invention can be greatly increased compared to the extract obtained by the extraction method previously described. More specifically, an azadirachtin content of 4% to 25% by weight in the extract of the present invention can be obtained, which is much higher than the corresponding content obtained in the prior art. In a preferred embodiment, the azadirachtin content in the extract of the present invention is at least 5% by weight, more preferably greater than 7% by weight. In a particularly preferred embodiment, the present invention achieves an azadirachtin content of 10% by weight or more in the extract. Therefore, according to the present invention, an efficient method for providing an extract of neem leaves having a high azadirachtin content is provided.

[0021] Furthermore, it has been found that the content of undesirable neem leaf components, in particular azadirachtin, can be reduced by the present invention. According to the present invention, the content of azadirachtin A and B in the extract is in particular less than 1% by weight and preferably less than 0.15% by weight. Other components such as Nimbin, typically in a relative amount of 0.1% to 0.6% by weight, may also be present, while most preferably, the extract of the present invention is completely free of detectable levels of azadirachtin A and B. Depending on the actual extraction conditions, the extract of the present invention may also include the following contents: substances selected from polyphenols, tannins and catechins (e.g., in an amount of 20% to 30% by weight), flavonoids (e.g., in an amount of 20% to 32% by weight), terpenes (e.g., in an amount of 10% to 20% by weight), pigments (e.g., in an amount of 15% to 25% by weight) and residual hydrocarbons present (e.g., in an amount of 10% to 20% by weight).

[0022] The neem leaf extract of the present invention is obtainable by the process of the present invention as described below.

[0023] As defined above, the process of the present invention for producing an extract of neem leaves comprises applying supercritical CO2 extraction under conditions favouring selective enrichment of the extract in azadirachtin content and / or selective reduction of azadirachtin content as compared to the average proportional contents of these components in neem leaves.

[0024] According to the present invention, supercritical CO2 extraction is carried out at a temperature of 50°C to 100°C.

[0025] According to a preferred embodiment of the invention, the method may also comprise the selective removal of one or more extract components other than azadirachtin, thereby again increasing the relative content of azadirachtin in the extract. In this context, for example, substances responsible for the mossy smell and / or green color of the extract may be removed by such additional selective removal by any technique applicable in this context. Such techniques are well known to the skilled person and include additional extraction methods and chromatographic methods such as, for example, flash chromatography.

[0026] The method of the present invention comprises harvesting neem leaves or providing neem leaves in an appropriate amount. Preferably, the neem leaves have not been treated with pesticides before or after being harvested, and leaves from organic farming are a preferred source of starting material for the method of the present invention. The neem leaves are washed and dried, and then crushed or ground and then subjected to supercritical CO2 extraction. The intensity of the crushing or grinding can be adjusted as required and is suitable for the extraction equipment and can be varied, for example, to obtain shredded leaves having the size of approximately oat flakes, but more finely ground and pulverized material can also be used.

[0027] In a preferred embodiment of the invention, the leaves are dried at temperatures up to 45°C and a preferred method for drying involves the use of air pressure.

[0028] The supercritical CO2 extraction is preferably carried out at a pressure of 200-600 bar, more specifically 250-350 bar. The temperature for supercritical CO2 extraction is 50-100°C and more specifically 55-85°C and especially 60-80°C.

[0029] The inventors have found that, despite the indications provided in the prior art literature that the use of temperatures above 45°C would damage the integrity of the plant components and that other groups have followed this indication in their extraction methods (e.g., US2020 / 0069636A1), the application of relatively high temperatures during the extraction process leads to an unexpected increase in the azadirachtin content in the extracts of the present invention. Compared to the prior art, in which temperatures below 45°C are applied, the present invention uses substantially elevated temperatures, which results in extremely high yields of intact azadirachtin and complete or almost complete elimination of azadirachtin.

[0030] The conditions of the process of the present invention further comprise using 5 to 30 kg CO2 / kg*h crushed or ground neem leaves, preferably 10 kg CO2 / kg*h neem leaves. According to a preferred embodiment of the present invention, supercritical CO2 extraction is carried out over a period of 1 to 5 hours, preferably 2 to 4 hours and most preferably 2,5 to 3,5 hours. Further preferably, during the entire extraction process, 20 to 40 kg CO2 / kg neem leaves are used for extraction, more preferably 25 to 35 kg CO2 / kg neem leaves and most preferably 30 kg CO2 / kg neem leaves.

[0031] As starting material for the process of the present invention, any neem leaves may be used. Preferably, neem leaves having an azadirachtin content of 0.01 wt-% up to 1 wt-%, preferably 0.05 wt-% up to 0.5 wt-% are employed.

[0032] The process of the present invention produces a highly purified neem extract containing a high content of azadirachtin and leads to very low content of azadirachtin or its complete elimination by applying only supercritical CO2 extraction and thereby allows avoiding any other solvents from being included in the extract. One of the advantages of the process of the present invention is that, due to the extraction process applied, the extract obtained does not contain the most commonly used extraction agents in the prior art processes, such as alcohol. However, for many applications, it is preferred or even necessary that alcohol is not present in the extract and the products obtained therefrom, such as in dermatological applications where the presence of alcohol may lead to dryness of the skin or other adverse skin reactions. Furthermore, the process of the present invention does not require any pretreatment of the dried and crushed or chopped leaf material as described in the prior art.

[0033] After the extraction has been carried out, the extract obtained may and preferably is heated to a temperature of about 40° C. to about 60° C. and subjected to a mixing step in order to improve the homogeneity of the extract obtained.

[0034] The process of the invention provides for the first time a one-step extraction process of crushed neem leaves which results in a highly satisfactory yield of azadirachtin in the extract obtained. The extract can be used directly in all known and conceivable applications of neem extracts and in particular azadirachtin. The extract has also been shown to be very well tolerated in any application form and formulation.

[0035] Thus, by the present invention, a high concentration of azadirachtin extracts can be provided at low cost in particular. Providing an extract with a high azadirachtin content allows the use of the extract in particular in cosmetic and pharmaceutical applications. In particular due to the high content of azadirachtin, only a relatively low proportion of the extract containing azadirachtin according to the present invention has to be added to a cosmetic or pharmaceutical composition.

[0036] A further subject of the present invention is a cosmetic and pharmaceutical composition comprising, respectively, an extract according to the invention or an extract obtained by the process according to the invention as described above.

[0037] The extract of the invention can be added directly to a cosmetic or pharmaceutical composition. In a preferred embodiment, the extract of the invention is first mixed with a standardizing agent. Therefore, the present invention further relates to a composition comprising (i) an extract of neem (Azadirachta indica) leaves as described herein and (ii) a standardizing agent, in particular caprylyl glycol and / or octyldodecanol.

[0038] In a preferred embodiment, the standardized composition is adjusted to the desired azadirachtin content. The preferred azadirachtin content of the standardized composition is 1 wt-% to 5 wt-% azadirachtin, in particular 2 wt-% to 4 wt-% azadirachtin. Thereupon, the extract of the invention is mixed with a standardizing agent preferably in an amount of 1:10 to 1:1, preferably 1:5 to 1:2 (weight extract / weight standardizing agent).

[0039] By mixing the extract according to the invention with a standardizing agent, it is possible in particular to adjust the viscosity of the extract.

[0040] The cosmetic composition of the present invention preferably contains the neem leaf extract of the present invention in a proportion of 0.1 wt-% to 5 wt-%, preferably 0.25 wt-% to 3 wt-%, and depending on the exact application form and intended indication of the cosmetic composition, the amount of the extract contained in the cosmetic composition may also be increased or decreased.

[0041] By using a standardized composition comprising the extract of the present invention and a standardizing agent, an amount of 0.1 to 5 wt-%, preferably 0.25 to 3 wt-% and more preferably 1 to 2.5 wt-% is included in the cosmetic composition.

[0042] Preferably, the cosmetic composition contains an azadirachtin content of at least 0.03 wt-%, more preferably at least 0.05 wt-% and more preferably at least 0.075 wt-%.Such high concentrations of azadirachtin in cosmetic products are obtained only because of the high content of azadirachtin in the extract of the present invention.

[0043] The cosmetic composition of the present invention further comprises a cosmetic base. Such a cosmetic base may include a water-in-oil emulsion, an oil-in-water emulsion, for example, a gel base, an ointment / cream base, a lotion base or any other form of cosmetic base. The extract of the present invention is soluble in oil, but insoluble in water. However, the addition of a suitable emulsifier also allows the use of the extract of the present invention in a water-based application form.

[0044] Depending on the intended treatment, the cosmetic composition according to the invention may also comprise other dermatologically active substances and / or, for example, analgesics.

[0045] For pharmaceutical compositions of the present invention, the content of the extract of the present invention in the composition is not limited. Pharmaceutical compositions usually contain auxiliary materials / excipients and / or carriers, and may also contain other additives for intended applications and formulations. Depending on the intended medical indication, the pharmaceutical active ingredient in the composition can be 100% neem extract of the present invention or a combination with other pharmaceutically active substances. For example, for use in dermatology, the pharmaceutical composition also preferably contains a suitable base for skin application, and for other medical applications, the extract can be used in pure form or in combination with other required or available substances. For some applications, formulations in the form of tablets, capsules, lozenges, or liquids for topical or oral administration or for injection are also further exemplary but non-restrictive dosage forms.

[0046] Applicable uses of the neem leaf extract of the present invention include all medical applications of neem products, and especially azadirachtin, which have been disclosed in the prior art.

[0047] Therefore, another subject of the present invention is the extract according to the invention or the pharmaceutical composition as described above for use in medicine, preferably in dermatology, in particular in the treatment of psoriasis, neurodermatitis, eczema and acne, in the treatment of cancer diseases and in the treatment of (other) inflammatory disorders.

[0048] The following examples and drawings further illustrate the present invention:

[0049] Figure 1 The reduction of oxidative stress due to the application of different concentrations of the neem leaf extract of the present invention in a skin model study is shown in comparison with a positive control and other active substances.

[0050] Figure 2 Additional skin model experiments are shown, and the anti-inflammatory effects of the neem leaf extract of the present invention at different concentrations compared to untreated controls. Example

[0051] Example 1: Preparation and analysis of neem leaf extract

[0052] The neem leaves are harvested, washed, and dried via air pressure drying at a temperature up to 45° C. After the drying process, the leaves are transferred to a hammer mill and ground into powder.

[0053] Supercritical CO2 extraction was carried out at 300 bar and 80°C at 30 m.rel (kg CO2 / kg leaf powder) for 3 hours. No additional extractants or pretreatments were applied. The extract obtained was heated to 40°C and mixed to achieve homogeneity.

[0054] The crude extract was standardized with caprylyl glycol, with the last standardization using a ratio of approximately 60% extract to 40% glycol.

[0055] Table 1 below shows HPLC analysis of several extract batches:

[0056] Table 1:

[0057]

[0058] This example shows that using the process of the present invention, an extract having a high azadirachtin content can be obtained even if the starting material is neem leaves having only a low content of azadirachtin.

[0059] Embodiment 2 (comparative example):

[0060] Commercially available neem leaf extract from Nisarga Biotech Private Limited (see US2020 / 0069636 A1) was purchased and analyzed.

[0061] The azadirachtin content of the extract was found to be 0.8 wt-%, and the amount of azadirachtin was found to be 0.003 wt-%.

[0062] Example 3: CO2 extraction at 60°C

[0063] The neem leaves are harvested, washed, and dried via air pressure drying at a temperature up to 45° C. After the drying process, the leaves are transferred to a hammer mill and ground into powder.

[0064] Supercritical CO2 extraction was carried out at 300 bar and 60°C at 30 m.rel (kg CO2 / kg leaf powder) for 3 hours. No additional extractants or pretreatments were applied. The extract obtained was heated to 40°C and mixed to achieve homogeneity.

[0065] The crude extract was standardized with caprylyl glycol, with the last standardization using a ratio of approximately 60% extract to 40% glycol.

[0066] Table 2 below shows the HPLC analysis of the crude extract:

[0067] Table 2:

[0068]

[0069] Example 4: In vitro studies on dermatological models

[0070] In vitro studies were conducted on a 3D skin model to evaluate the antioxidant potential and anti-inflammatory properties of the neem leaf extract of the present invention and skin barrier regeneration after administration of the extract.

[0071] The neem leaf extract was obtained at 300 bar and 80° C. as described in Example 1. The neem leaf extract was standardized to 3 wt-% azadirachtin content using caprylyl glycol.

[0072] In the tests conducted, standardized neem leaf extract was used in concentrations ranging from 0.5% to 2.5% and compared to a positive control of untreated skin models and 2.5% vitamin C. A further comparison was made with the application of 0.01% astaxanthin, which is often used as the "gold standard" of antioxidant substances.

[0073] This study revealed a significant reduction in oxidative cellular stress. Even the smallest dose of the extract, 0.5%, showed significant improvement over vitamin C administration and over 90% reduction in cellular stress. The extract at a concentration of 2.5% had about the same effect as astaxanthin on cellular stress reduction ( Figure 1 ).

[0074] Furthermore, the present study shows a significant anti-inflammatory effect of the extract according to the invention. The skin model was treated with SLS on day 1. Immediately after this SLS treatment, the extract according to the invention was applied to the skin model. The extract-treated skin model showed significantly lower IL-6 concentrations than the untreated sample. Already on day 3, when using 2,5% of the extract, a further significant reduction in IL-6 concentrations was detected. Treatment with 1% of the extract showed a corresponding reduction on day 4 ( Figure 2 ).

[0075] Skin model studies clearly show the significant beneficial effects of the neem leaf extract of the present invention even at low concentrations for both its antioxidant and anti-inflammatory properties.

[0076] Example 5: Wound healing

[0077] In patient tissues damaged by radiation during cancer therapy, treated with a pharmaceutical composition comprising the neem leaf extract of the present invention in an amount resulting in 0.03 wt-% azadirachtin in the pharmaceutical composition. Thus, rapid wound healing was observed. It is presumed that the antioxidant and anti-inflammatory effects of the extract containing azadirachtin of the present invention promote rapid wound healing.

Claims

1. An extract of neem (Azadirachta indica) leaves, said extract being enriched in azadirachtin content as compared to the relative average proportion of azadirachtin content in neem leaves, said extract containing 4 to 25 wt. % of azadirachtin.

2. The extract of neem leaves as claimed in claim 1, containing 4 to 25 wt% azadirachtin, preferably at least 5 wt% and more preferably 10 to 20 wt% azadirachtin.

3. An extract of neem leaves as claimed in claim 1 or 2, containing less than 1 wt. %, preferably less than 0.15 wt. % azadirachtin.

4. A process for producing an extract of neem leaves according to any one of claims 1 to 3, wherein the process comprises applying supercritical CO2 extraction under conditions conducive for selective enrichment of the azadirachtin content in the extract as compared to the average proportional content of azadirachtin in neem leaves, wherein the supercritical CO2 extraction is carried out at a temperature of 50°C to 100°C.

5. The method of claim 4, wherein the proportion of azadirachtin in the extract obtained by supercritical CO2 extraction is further increased by selectively removing one or more other extract components.

6. A process as claimed in claim 4 or 5, wherein neem leaves are harvested, washed, dried and crushed or ground before supercritical CO2 extraction.

7. The method of claim 6, wherein drying is performed at a temperature up to 45°C, preferably via air pressure drying.

8. The method of any one of claims 4 to 7, wherein the supercritical CO2 extraction is carried out at a pressure of 200 to 600 bar, preferably 250 to 350 bar and / or at a temperature of 55 to 85°C and preferably 60 to 80°C.

9. The method of any one of claims 4 to 8, wherein the supercritical CO2 extraction is performed for 1 to 5 hours, preferably 2 to 4 hours and most preferably 2.5 to 3.5 hours.

10. The process according to any one of claims 4 to 10, wherein the supercritical CO2 extraction is carried out using 20 to 40 kg CO2 / kg crushed or ground neem leaves, preferably 25 to 35 kg CO2 / kg, and / or wherein the supercritical CO2 extraction is carried out using about 10 kg CO2 per kg neem leaves and per hour of extraction time.

11. A composition comprising: (i) an extract of neem (Azadirachta indica) leaves according to any one of claims 1 to 3 or obtained by the process according to any one of claims 4 to 10, and (ii) Standardizing agents, in particular caprylyl glycol and / or octyldodecanol.

12. A cosmetic composition comprising the extract according to any one of claims 1 to 3 or the extract obtained by the method according to any one of claims 4 to 10, or the composition according to claim 11.

13. The cosmetic composition of claim 12, wherein the extract or composition is contained in an amount of 0.1 wt-% to 5 wt-%, preferably 0.25 wt-% to 3 wt-%, based on the total weight of the cosmetic composition.

14. A pharmaceutical composition comprising the extract according to any one of claims 1 to 4 or the extract obtained by the method according to any one of claims 4 to 10, or the composition according to claim 11.

15. The extract according to any one of claims 1 to 3 or the composition according to claim 11 or the cosmetic or pharmaceutical composition according to any one of claims 12 to 14 for use in dermatology, in particular in the treatment of psoriasis, in the treatment of cancer diseases or in the treatment of inflammatory disorders.

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