Neem tree leaf extract

Abstract

The neem tree leaf extract of the present invention is enriched in nimbolide and / or depleted in azadirachtin compared to the relative average proportional contents of these components in neem tree leaves. A method for producing such an extract of the present invention comprises applying supercritical CO2 extraction under conditions favoring selective enrichment of nimbolide and / or selective depletion of azadirachtin in the extract.

Description

[Technical Field]

[0001] explanation The present invention relates to a neem tree (Azadirachta indica) leaf extract, which is rich in a specific component of neem tree leaves, namely nimbolide. The present invention also relates to a method for obtaining the extract of the present invention, as well as cosmetic and pharmaceutical compositions containing such extract. The present invention also relates to the neem leaf extract of the present invention for use in skin care and dermatology, as well as other medical fields, which can be used to treat and alleviate diseases and their symptoms. [Background technology]

[0002] Background and Prior Art For thousands of years, the neem tree (Azadirachta indica) has been known as a medicinal plant. It is particularly popular in rural India, where it is often referred to as the "village pharmacy," but it is also widely used in other parts of the world. Plant parts, such as leaves, bark, and seeds, and products derived from them are used for a variety of purposes and are well established as Ayurvedic medicinal repositories. Many of the uses of neem tree products are based on the traditional knowledge of people in countries where the tree has grown for centuries.

[0003] Various plant parts are believed to serve different, but partly the same, or at least overlapping, purposes. For example, neem oil, obtained from the seeds of the neem tree, is best known for its use as an insecticide, but is also used in cosmetics and as a natural spermicide. However, oral consumption of neem oil is not recommended. Neem tree leaves are widely used in the cosmetic industry as well as for medicinal purposes.

[0004] In the Western Hemisphere, scientific investigations were initiated by the work of Professor Schmutterer (see, for example, Heinrich Schmutterer (Hrsg.): The Neem Tree, Azadirachta indica (A. Juss.) and Other Melia Plants: A Source of Unique Natural Products for Integrated Pest Management, Medical, Industrial and Other Purposes. VCH Verlagsgesellschaft, Weinheim 1995, ISBN 3-527-30054-6). He investigated the use of neem tree products as insecticides after discovering that the neem tree was the only plant to survive a particularly devastating locust plague. His research focused primarily on azadirachtin and other plant compounds that exhibit insecticidal properties.

[0005] Truly scientific literature on ingredients, their isolation, and their uses and application forms is very limited and inconclusive. The studies documented in such literature often do not adhere to scientific standards, and the results reported therein are often not reproducible. Also, reliable analyses for comparing observed effects are often lacking. For example, the origin of neem leaves is often listed as "from our university garden," and the actual content of these ingredients in this raw material has never been analyzed, or at least not in a reproducible manner.

[0006] In addition to its use in traditional medicine, neem and certain components that can be enriched or isolated from the plant have also been investigated in recent years for their application in the treatment of various diseases, particularly cancer. Studies have shown surprising benefits from the application of such substances, and it is speculated that the anti-cancer effects are due to the modulation of multiple cellular processes by the application of neem extracts or purified or isolated neem components.

[0007] One notable constituent is nimbolide, which has shown activity against many different types of cancer in in vivo and in vitro studies. Nimbolide has also been shown to induce apoptosis while inhibiting metastasis and angiogenesis. Other constituents of the neem tree have been shown to have anti-inflammatory and other medically relevant properties.

[0008] The prior art describes various methods for obtaining extracts containing various neem components. For example, US 2020 / 0069636 A1 summarizes several relevant prior art documents and the methods described therein, including extraction methods using specific solvents and solvent combinations, as well as partial precipitation and chromatographic fractionation. Relevant documents include WO 2015 / 035199 A1, CN 101972246 B, CN 103864876 A, US 5,370,873 A, JP 2009 / 274956 A, and WO 2007 / 137389 A1. However, these methods are considered unsuitable because they use relatively high temperatures for separation, which are believed to destroy or alter the molecular structure of temperature-sensitive components with therapeutic value. Furthermore, the use of solvents is considered undesirable due to potential adverse effects on human health and, further, the possibility that the solvents themselves may be carcinogenic.

[0009] The authors of US 2020 / 0069636 A1 propose a process for preparing a CO2 extract of neem, in which heat-sensitive plant components are obtained during the extraction process by applying supercritical CO2 extraction conditions at temperatures below 45°C, which are known not to adversely affect heat-sensitive plant components. However, the method requires many process steps, including repeated extractions. More specifically, in addition to the supercritical CO2 extraction, an additional extraction is performed using a mixture of CO2 and ethyl alcohol, and the method requires vacuum distillation to remove the solvent. Thus, the process disclosed in this document requires fairly sophisticated procedures, and the use of solvents cannot yet be completely eliminated to achieve a satisfactory yield of the desired neem components.

[0010] In the context of research into the medical use of nimbolide for various diseases and conditions, the use of commercially available nimbolide may be considered. However, particularly in cases involving diseases that are not life-threatening but require symptomatic relief and treatment, the use of commercially available nimbolide is not a preferred option because even small amounts of this substance are very expensive. Furthermore, in such cases, the synergistic effects provided by the presence of other neem components are lost.

[0011] For many applications, particularly certain medical uses, some neem components, such as azadirachtin, are considered undesirable because they can have adverse effects on the human body when applied or ingested, especially over long periods of time. Also, for dermatological purposes, the inclusion of such substances is undesirable.

[0012] Therefore, the objectives underlying the present invention are, on the one hand, to provide a neem extract containing a higher concentration of nimbolide compared to known extracts, and, on the other hand, to provide a simple and cost-effective method for producing such an extract while avoiding the extraction process as well as contamination with undesirable substances from the neem plant. Summary of the Invention [Means for solving the problem]

[0013] Brief description of the invention This object is solved as a first aspect of the present invention by providing an extract of neem tree (Azadirachta indica) leaves, which extract is enriched in nimbolide content as compared to the relative average proportional content of this component in neem tree leaves, the extract containing 4 to 25% by weight of nimbolide.

[0014] In a second aspect of the present invention, there is provided a method for producing the inventive extract of neem tree leaves, the method comprising applying supercritical CO2 extraction under conditions favourable to selectively enrich the nimbolide content in the extract compared to the average proportional content of this component in neem tree leaves, wherein the supercritical CO2 extraction is carried out at a temperature of 50°C to 100°C.

[0015] In the third and fourth aspects of the present invention, cosmetic and pharmaceutical compositions are provided comprising the extract of the present invention as defined above. In the fifth aspect of the present invention, the extract of the present invention and the cosmetic or pharmaceutical compositions of the present invention are provided for use in dermatology, the treatment of cancer diseases and the treatment of inflammatory conditions.

[0016] Detailed Description of the Invention Within the context of their research, the inventors have been able to establish a method that allows for highly efficient and selective extraction of nimbolide and other constituents of neem tree leaves.

[0017] In particular, the nimbolide content in the neem extract of the present invention can be significantly improved compared to extracts obtained by previously described extraction methods. More specifically, a nimbolide content of 4 to 25% by weight can be obtained in the extract of the present invention, which is much higher than the respective contents obtained by the prior art. In a preferred embodiment, the nimbolide content in the extract of the present invention is at least 5% by weight, more preferably 7% by weight or more. In a particularly preferred embodiment, the present invention achieves a nimbolide content of 10% by weight or more in the extract. Thus, the present invention provides an efficient method for obtaining a neem leaf extract having a high nimbolide content.

[0018] Furthermore, it has been found that the present invention can reduce the content of undesirable components in neem tree leaves, particularly azadirachtin. According to the present invention, the content of azadirachtins A and B in the extract is less than 1% by weight, preferably less than 0.15% by weight. Other components, such as nimbin, may also be present, typically in relative amounts of 0.1-0.6% by weight. Most preferably, the extract of the present invention contains no detectable levels of azadirachtins A and B. Depending on the actual extraction conditions, the extract of the present invention may also contain substances selected from polyphenols, tannins, and catechins, for example, in amounts of 20-30% by weight; flavonoids, for example, in amounts of 20-32% by weight; terpenoids, for example, in amounts of 10-20% by weight; pigments, for example, in amounts of 15-25% by weight; and residual hydrocarbons, for example, in amounts of 10-20% by weight.

[0019] The neem leaf extract of the present invention can be obtained by the method described below.

[0020] The present method for producing the above defined neem tree leaf extract comprises applying supercritical CO2 extraction under conditions favouring selective enrichment of the nimbolide content and / or selective depletion of the azadirachtin content in the extract compared to the average proportional content of these components in the neem tree leaves.

[0021] According to the present invention, the supercritical CO2 extraction is carried out at a temperature of 50°C to 100°C.

[0022] According to a preferred embodiment of the present invention, the method may also include the selective removal of one or more extract components other than nimbolide, thus again increasing the relative content of nimbolide in the extract. In this context, for example, substances responsible for the mossy odor and / or greenish color of the extract may be removed by such additional selective removal carried out by any technique applicable in this context. Such techniques are well known to those skilled in the art and include further extraction methods and chromatographic methods, such as flash chromatography.

[0023] The method involves harvesting neem leaves or providing a suitable amount of neem leaves. Preferably, the neem leaves have not been treated with pesticides before or after harvest, and leaves from organic farming are the preferred source of starting material for the method of the present invention. The neem leaves are washed and dried and then crushed or ground before being subjected to supercritical CO2 extraction. The intensity of the crushing or grinding can be adapted as necessary and appropriate for the extraction plant and can be varied, for example, to obtain crushed leaves of the size of rolled oats, although more finely crushed and ground material can also be used.

[0024] 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 an air press.

[0025] The supercritical CO2 extraction is preferably carried out at a pressure of 200 to 600 bar, more particularly 250 to 350 bar. The temperature of the supercritical CO2 extraction is 50 to 100°C, more particularly 55 to 85°C, in particular 60 to 80°C.

[0026] The present inventors have discovered that applying relatively high temperatures in the extraction process leads to an unexpected increase in nimbolide content in the extract of the present invention, despite suggestions provided in prior art literature and adhered to by other groups regarding their extraction methods (e.g., US 2020 / 0069636 A1) that using temperatures above 45°C would compromise the integrity of the plant components. In contrast to the prior art, in which temperatures below 45°C were applied, the present invention uses significantly higher temperatures, resulting in an exceptionally high yield of intact nimbolide and complete or near-complete removal of azadirachtin.

[0027] The process conditions of the present invention further include the use of 5 to 30 kg of CO2 per kg of crushed or ground neem leaves per hour (5 to 30 kg CO2 / kg*h crushed or ground neem leaves), preferably 10 kg of CO2 per kg of neem leaves per hour (10 kg CO2 / kg*h neem leaves). According to a preferred embodiment of the present invention, the supercritical CO2 extraction is carried out for a period of 1 to 5 hours, preferably 2 to 4 hours, and most preferably 2.5 to 3.5 hours. More preferably, 20 to 40 kg of CO2 per kg of neem leaves is used for the extraction during the entire extraction process, more preferably 25 to 35 kg of CO2 per kg of neem leaves, and most preferably 30 kg of CO2 per kg of neem leaves.

[0028] Any neem tree leaves can be used as starting material for this process, preferably neem tree leaves having a nimbolide content of 0.01 to 1 wt%, more preferably 0.05 to 0.5 wt%.

[0029] By applying only supercritical CO2 extraction, the present method produces a highly purified neem extract containing a high content of nimbolide and an extremely low or completely eliminated azadirachtin content, thus avoiding the inclusion of any other solvents in the extract. One advantage of the present method is that, depending on the extraction method used, the resulting extract does not contain extractants such as alcohol, which are most commonly used in prior art methods. However, in many applications, such as dermatological applications where the presence of alcohol can cause dryness or other adverse skin reactions, it is preferable or even essential that the extract and products derived therefrom be alcohol-free. Furthermore, the present method does not require any pretreatment of the dried and crushed or shredded leaf material, as described in the prior art.

[0030] After the extraction, in order to improve the homogeneity of the obtained extract, it is possible and preferable to warm the obtained extract to a temperature of about 40°C to about 60°C and carry out a mixing step.

[0031] The present method provides for the first time a single step extraction process for crushed neem leaves which results in a very satisfactory yield of nimbolide in the extract obtained. This extract can be directly used in all known and possible applications of neem extract, particularly nimbolide. Also, this extract has been shown to be very well tolerated in all applications and formulations.

[0032] Therefore, the present invention makes it possible to provide a particularly highly concentrated nimbolide extract at low cost. The provision of an extract with a high nimbolide content enables the use of this extract in particular in cosmetic and pharmaceutical applications. In particular, due to the high nimbolide content, only a relatively low proportion of the nimbolide-containing extract of the present invention needs to be added to a cosmetic or pharmaceutical composition.

[0033] Further subject matter of the present invention are cosmetic and pharmaceutical compositions comprising, respectively, the extract according to the invention or the extract obtainable by the method according to the invention described above.

[0034] The extract of the present invention can be added directly to a cosmetic or pharmaceutical composition. In a preferred embodiment, the extract of the present invention is first mixed with a standardizing agent. Thus, the present invention further relates to a composition comprising (i) a neem tree (Azadirachta indica) leaf extract as described herein, and (ii) a standardizing agent, in particular caprylyl glycol and / or octyldodecanol.

[0035] In a preferred embodiment, the standardized composition is adjusted to the desired nimbolide content. A preferred nimbolide content of the standardized composition is 1-5 wt. % nimbolide, particularly 2-4 wt. % nimbolide. Therein, the extract of the present invention is mixed with a standardizer in an amount (weight of extract / weight of standardizer) of preferably 1:10 to 1:1, preferably 1:5 to 1:2.

[0036] The extract of the invention can be mixed with a standardizing agent, in particular to adjust the viscosity of the extract.

[0037] The cosmetic composition of the present invention preferably contains the neem leaf extract of the present invention in a proportion of 0.1 to 5% by weight, preferably 0.25 to 3% by weight, although the amount of extract contained in the cosmetic composition may be increased or decreased depending on the exact form of application and the intended indication of the cosmetic composition.

[0038] By using a standardized composition containing the extract of the present invention and a standardizing agent, the cosmetic composition contains the extract in an amount of 0.1 to 5 wt%, preferably 0.25 to 3 wt%, more preferably 1 to 2.5 wt%.

[0039] Preferably, the cosmetic composition contains a nimbolide content of at least 0.03 wt-%, more preferably at least 0.05 wt-%, even more preferably at least 0.075 wt-%. Obtaining such high nimbolide concentrations in cosmetic products is only possible due to the high content of nimbolide in the extract of the present invention.

[0040] The cosmetic composition of the present invention further comprises a cosmetic base. Such cosmetic bases can include water-in-oil emulsions, oil-in-water emulsions, such as gel bases, ointment / cream bases, lotion bases, or any other form of cosmetic base. The extract of the present invention is easily soluble in oil but insoluble in water. However, by adding an appropriate emulsifier, the extract of the present invention can also be used in aqueous application forms.

[0041] Depending on the intended treatment, the cosmetic compositions of the invention may also contain other dermatologically active substances and / or pain relievers, for example.

[0042] In the case of 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 substances / excipients and / or carriers, and optionally further additives for the intended use and formulation. The pharmaceutically active ingredient in the composition can be 100% neem extract of the present invention or a combination with other pharmaceutically active substances, depending on the intended medical indication. For example, in the case of dermatological use, the pharmaceutical composition preferably contains a suitable base for skin application. For other medical uses, the extract can be used in pure form or in combination with additional necessary or useful substances. For certain uses, formulations as tablets, capsules, lozenges, or liquid forms for topical or oral administration or injection are further exemplary, but not limiting, dosage forms.

[0043] The applicable uses of the neem leaf extract of the present invention include all the medicinal uses of neem products, particularly nimbolide, already disclosed in the prior art.

[0044] Another subject of the present invention is therefore an extract according to the invention or a 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 conditions.

[0045] The present invention is further illustrated by the following examples and figures: [Brief explanation of the drawings]

[0046] [Figure 1] FIG. 1 shows the reduction in oxidative stress as a result of application of different concentrations of the neem leaf extract of the present invention compared to a positive control and other actives in a skin model study. [Figure 2] Figure 2 shows further skin model experiments and the anti-inflammatory effect of different concentrations of the neem leaf extract of the present invention compared to an untreated control.

[0047] Example Example 1: Preparation and Analysis of Neem Tree Leaf Extract Neem tree leaves were harvested, washed and dried by air drying at temperatures up to 45° C. After the drying process, the leaves were transferred to a hammer mill and crushed into a powder.

[0048] Supercritical CO2 extraction was performed at 300 bar and 80°C for 3 hours at 30 mrel (kg CO2 / kg leaf powder). No further extractant or pretreatment was used. The resulting extract was heated to 40°C and mixed to homogeneity.

[0049] The raw extract was standardized with caprylyl glycol, and the final standardization used a ratio of approximately 60% extract to 40% glycol.

[0050] Table 1 below shows the HPLC analysis of several extraction batches:

[0051] [Table 1]

[0052] This example shows that even when neem leaves having a low nimbolide content are used as a starting material, an extract having a high nimbolide content can be obtained by using the method of the present invention.

[0053] Example 2 (Comparative Example): Commercially available neem leaf extract from Nisarga Biotech Private Limited (Reference: US 2020 / 0069636 A1) was purchased and analyzed.

[0054] The nimbolide content of the extract was found to be 0.8 wt% and the azadirachtin content was found to be 0.003 wt%.

[0055] Example 3: CO2 extraction at 60°C Neem tree leaves were harvested, washed and dried by air drying at temperatures up to 45° C. After the drying process, the leaves were transferred to a hammer mill and crushed into a powder.

[0056] Supercritical CO2 extraction was performed at 300 bar and 60°C for 3 h at 30 mrel (kg CO2 / kg leaf powder). No further extractant or pretreatment was used. The resulting extract was heated to 40°C and mixed to homogenize.

[0057] The raw extract was standardized with caprylyl glycol, and the final standardization used a ratio of approximately 60% extract to 40% glycol.

[0058] Table 2 below shows the HPLC analysis of the raw extract:

[0059] [Table 2]

[0060] Example 4: In vitro studies on skin models An in vitro study was carried out on a 3D skin model to evaluate the antioxidant and anti-inflammatory properties of the neem tree leaf extract of the present invention and skin barrier regeneration after application of the extract.

[0061] Neem leaf extract was obtained at 300 bar and 80° C. as described in Example 1. The neem leaf extract was standardized to a nimbolide content of 3 wt-% with caprylyl glycol.

[0062] The studies performed used standardized neem leaf extract at concentrations between 0.5% and 2.5% and compared it to a positive control of an untreated skin model and 2.5% vitamin C. Further comparisons were made with the application of 0.01% astaxanthin, which is commonly used as the "gold standard" for antioxidants.

[0063] The study revealed a significant reduction in oxidative cellular stress. Even the lowest dose of 0.5% extract showed a significant improvement over application of vitamin C and reduced cellular stress by more than 90%. A 2.5% concentration of the extract had almost the same effect as astaxanthin in reducing cellular stress (Figure 1).

[0064] Furthermore, this study demonstrates the significant anti-inflammatory effect of the extract of the present invention. The skin model was treated with SLS on day 1. Immediately after this SLS treatment, the extract of the present invention was applied to the skin model. The skin model treated with the extract showed significantly lower IL-6 concentrations than the untreated sample. Already on day 3, a further clear decrease in IL-6 concentration was detected when using 2.5% extract. Treatment with 1% extract showed a comparable decrease on day 4 (Figure 2).

[0065] Skin model studies clearly show that the neem leaf extract of the present invention, even at low concentrations, has significant beneficial effects in both its antioxidant and anti-inflammatory properties.

[0066] Example 5: Wound healing When patient tissues damaged by radiation during cancer treatment were treated with a pharmaceutical composition containing the neem leaf extract of the present invention, the amount of nimbolide in the pharmaceutical composition was 0.03 wt%. As a result, rapid wound healing was observed. It is presumed that this rapid wound healing is promoted by the antioxidant and anti-inflammatory effects of the nimbolide-containing extract of the present invention.

Claims

1. An extract of neem tree (Azadirachta indica) leaves, wherein the nimbolide content is enriched compared to the relative average proportional content of this component in neem tree leaves, and the extract contains 4 to 25% by weight of nimbolide.

2. 2. The neem tree leaf extract according to claim 1, containing 4 to 25% by weight of nimbolide, preferably at least 5% by weight and more preferably 10 to 20% by weight of nimbolide.

3. 3. An extract of neem tree leaves according to claim 1 or 2 containing less than 1% by weight, preferably less than 0.15% by weight of azadirachtin.

4. 4. A method for producing the neem tree leaf extract according to any one of claims 1 to 3, comprising the step of: subjecting the extract to supercritical CO 2 treatment under conditions favourable for selectively enriching the nimbolide content in the extract relative to the average proportional content of this component in the neem tree leaves. 2 wherein the extraction comprises applying supercritical CO 2 A method wherein the extraction is carried out at a temperature of 50°C to 100°C.

5. supercritical CO 2 5. The method of claim 4, wherein the proportional content of nimbolide in the extract obtained by extraction is further increased by selective removal of one or more other extract components.

6. Neem leaves are grown in supercritical CO 2 6. The method according to claim 4 or 5, wherein the plant is harvested, washed, dried and crushed or ground prior to extraction.

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

8. supercritical CO 2 8. The method according to any one of claims 4 to 7, wherein the 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, preferably 60 to 80°C.

9. supercritical CO 2 9. The method according to any one of claims 4 to 8, wherein the extraction is carried out for 1 to 5 hours, preferably 2 to 4 hours, most preferably 2.5 to 3.5 hours.

10. supercritical CO 2 Extraction requires 20-40 kg of CO per kg of crushed or ground neem leaves. 2 , preferably 25-35 kg CO per kg 2 and / or supercritical CO 2 Extraction requires approximately 10 kg of CO per kg of neem leaves and per hour of extraction time. 2 The method according to any one of claims 4 to 10, which is carried out using

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

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. 13. The cosmetic composition according to claim 12, wherein the extract or composition is contained in an amount of 0.1 to 5 wt %, preferably 0.25 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. An extract according to any one of claims 1 to 3, or a composition according to claim 11, or a 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 conditions.

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