HIGH PURITY ALPHA YOHIMBINE (RAUWOLSCINE) FROM RAUWOLFIA SPECIES

Abstract

Present invention discloses a novel, commercially viable process for extraction of Alpha yohimbine from the bark, stem and leaves of Rauwolfia species. The extract is obtained by a precipitation method involving alternate steps of acidification and alkalization along with use of specific organic solvents. The extract gives higher yield of 7-8 fold as compared to other processes and without use of any column chromatography at all. Yield of alpha yohimbine from roots and leaves of Rauwolfia canescens by process of present invention was 0.017% and 0.4% respectively, indicating that leaves are a much better source. The HPLC analysis of the compound obtained indicated a purity of >90%.

Claims

1. Alpha yohimbine having a purity of at least 90% obtained from the leaves of Rauwolfia canescens.

2. Alpha yohimbine as claimed in claim 1 having a purity of 90-96%.

Description

DESCRIPTION OF THE DRAWINGS

[0048] FIG. 1: Structure of Yohimbine.

[0049] FIG. 2: Structure of Alpha-Yohimbine (Rauwolscine).

[0050] FIG. 3: HPLC analysis to determine the purity.

DETAILED DESCRIPTION OF THE INVENTION

[0051] Present invention discloses a novel, commercially viable process for extraction of Alpha yohimbine from the leaves of Rauwolfia species though bark and stem can also be used. The advantage of using leaves is that it represents a renewable source which is easily available and easy to handle and process. Secondly, the improved process of present invention increases the yield of alpha yohimbine by 7-8 fold which is quite remarkable. Unlike prior an methods which suffer from the limitations of very poor yield and low purity of the compound obtained, the method of present invention yields the desired compound i.e. Alpha yohimbine in high quantities (upto 7-8 fold increase over existing methods) and in high purity (95% vs 39% in a prior an method using leaves of Rauwolfia species. Comparison of extraction process of alpha yohimbine from leaves of Rauwolfia species with a prior an process (US 20120184576 A1) is given in Table 1 above.

[0052] Challenges in extraction of pure molecules from plant materialExtraction of desired molecules front plant sources poses several challenges, especially low yields and tow impurities. The extract from plant material e.g. root, stem, leaf, bark, fruit, flower, seeds or whole plant is often contaminated with undesirable impurities which lower the commercial value of the product besides posing health risks due to presence of undesirable impurities. Hence, it is highly desirable to develop suitable processes/methods to obtain desired molecules of high purity and in high quantity.

[0053] Existing approaches for obtaining desired molecules from plant products and their limitationsPlants are a rich source of various chemicals and compounds. These are commonly referred to as Phytochemicals i.e. chemicals derived from plants. Depending upon the nature of atoms present and also types of chemical groups present in the compounds/molecules, the molecules differ in size and polarity. These differences in size and polarity form the basis of various separation techniques e.g. chromatography, solvent extraction and also acidification/alkalization which are commonly used at commercial level to extract desired molecules from plants.

[0054] LimitationGel permeation and ion-exchange chromatography exploit differences of size and charge of the molecules respectively to bring about separation and purification of the molecules. However, they suffer from the limitation of being extremely slow, cumbersome and also use of expensive matrices (packing material inside the column). The yield is also quite low though purity of the desired molecules may be high Solvent extraction offers the benefit of low-cost, high yield but suffers from the limitation of low purity of tic molecules obtained.

[0055] From the above it is clear that no process is available in the prior art for production of alpha yohimbine at commercial scale and high purity level. Both these challenges viz. commercial/industrial scale production and high level of purity of alpha yohimbine have been overcome by the process of present invention which is simple, economical and results in high yields of alpha yohimbine with high purity levels of between 90-96%.

[0056] Theoretical Concepts involved in Process of Present Invention

[0057] To ensure better understanding of the process of he invention, the theoretical aspects involved are discussed. It is a known fact in chemistry that like dissolves like i.e. a solvent which is polar will dissolve a compound which is polar and not a compound which is non-polar e.g. water is polar and oil is non polar. Hence, water will not dissolve oil since nature of both are entirely different i.e. polar and non-polar will not mix. Plants contain i number of compounds and depending upon their composition of atoms and arrangement of groups, the compounds may be polar, non-polar, strongly polar/weakly polar etc. Hence, when extraction is carried out with non-polar and polar solvents, depending upon their polarity, the compounds get extracted into the appropriate phase e.g. highly polar compounds get extracted with water, while non-polar are not extracted. The polarity of a solvent or a compound depends upon the number of polar groups present in it. Polar value of some of the commonly used solvents for preparation of plant extracts is given in Table 2 below;

TABLE-US-00002 TABLE 2 Polarity of some commonly used solvents in plant extracts S. Polar No. Non-Polar Solvent Polar Value Polar Solvent Value 1. Hexane, Benzene 0.0 Ethyl acetate 5.3 2. Toluene 1.4 Dichloromethane 7.3 3 Diethyl ether 2.9 Acetone 10.4 4. Chloroform 3.1 Acetonitrile 18.0 5. 1,4-Dioxane 1.8 Water 16.0 Data Source: https://en.wikipedia.org/wiki/Solvent

[0058] When a solvent or a mixture of solvents is added to a plant material, it will draw out the chemical compounds which are of like or similar nature i.e. polar solvents will extract polar compounds and non-polar compounds will be extracted by ion-polar solvents. Solvents of similar polarity to that of die compounds being extracted will be more suitable than those whose polarity is different.

[0059] Thus one can extract different compounds at different steps of extraction by using a particular solvent or a mixture of solvents. However, lass of some quantity of the desired compound invariably occurs when number of extraction steps is more, leading to low yields. However, more steps of extraction with different solvents result in high purity compounds. When number of steps of extraction are reduced, yield increases but purity decreases.

[0060] One way to overcome this problem is to separate the extraction and purification steps Extraction is carried out using organic solvents which gives good yields but desired compound is of low purity. However, if high purity compound is required, then purification is carried out using suitable chromatographic techniques e.g. column chromatography.

[0061] Extraction of Compounds using Organic Solvents along with Acidification/Alkalization

[0062] Apart from polarity, another factor affecting solubility of plant compounds and hence extraction is pH i.e. the acidic or basic nature of the solvent. By addition of an acid or a base to an organic solvent, its properties can be drastically altered and separation of compounds can be achieved. The concept is further elaborated below:

[0063] Compounds (Salts), which are ionic, tend to be water-soluble while neutral molecules tend not to be. The addition of an acid to a mixture of an organic base and acid will result in the acid remaining uncharged, while the base will be protonated to form a salt. If the organic acid, such as a carboxylic acid, is sufficiently strong, its self-ionization can be suppressed by the added acid. Conversely, the addition of a base to a mixture of an organic acid and base will result in the base remaining uncharged, while the acid is deprotonated to give die corresponding salt. Once again, the self-ionization of a strong base is suppressed by the added base. The acid-base extraction procedure can also be used to separate very weak acids from stronger acids and very weak bases from stronger bases, as long as the difference of their pKa (or pKb) constants is large enough e.g. weak acids with phenolic OH groups like phenol, 2-naphthol, or 4-hydroxyindole (pKa around 10) from stronger acids like benzoic acid or sorbic acid (pKa around 4-5); very weak bases like caffeine or 4-nitroaniline (pKb around 13-14) from stronger bases like mescaline or dimethyltryptamine (pKb around 3-4).

[0064] Usually the pH is adjusted to a value roughly between the pKa (or pKb) constants of the compounds to be separated. Weak acids like citric acid, phosphoric acid, or diluted sulfuric acid are used for moderately acidic pH values, and hydrochloric acid or more concentrated sulfuric acid is used for strongly acidic pH values. Similarly, weak bases like ammonia or sodium bicarbonate (NaHCO.sub.3) are used for moderately basic pH valves while stronger bases like potassium carbonate (K.sub.2CO.sub.3) or sodium hydroxide (NaOH) are used for strongly alkaline conditions. In present case, the pKa value of the target compound i.e. alpha yohimbine is 6.34. Optimization of various solvents and acidification steps was carried out by inventors resulting in a novel process which gave high yields and desired purity of alpha yohimbine.

[0065] Methodology

[0066] The process comprises of the following 7 steps: [0067] 1. Organic solvent-Alkaline extraction step: Appropriate part of Rauwolfia sp. viz. bark, stem, root etc. in dried and powdered form is extracted multiple times, optimally 3 times, with a water-immiscible, non-polar organic solvent preferably toluene, at 40-50 C. in ratio of 1:4 i.e. 1 part solid and 4 parts solvent and made alkaline (pH 9-9.5) by addition of alkali, preferably 20-25% solution of ammonia and keeping for 4 hours in reactor with stirring followed by filtration. The filtrates extracts are then pooled. [0068] 2. Organic solvent-Acidic extraction step: The pooled organic solvent extracts are then made acidic (pH 3.50.5) by addition of acid solution, preferably tartaric acid and extracted multiple times, optimally 3 times, with purified water in optimal ratio of 12:1 v/v (volume of pooled fraction, volume of water), to extract alpha yohimbine (rauwolscine) into acidified aqueous layer with high efficiency. [0069] 3. Alkalinisation of aqueous layer: The acidified aqueous layer containing alpha yohimbine (rauwolscine) is then made alkaline (pH 9.0-9.5) and extracted multiple times, optimally 3 times with chlorinated, non-polar solvent such as chloroform (4:1 v/v; volume of aqueous pooled fraction: volume of solvent) to extract alpha yohimbine (rauwolscine) into the organic layer and leave impurities behind in the basic aqueous layer. [0070] 4. Recovery of alpha yohimbine from organic solvent by evaporation of solvent: Alpha yohimbine (rauwolscine) is obtained as dry residue from the organic layer by distillation to evaporate the solvent. [0071] 5. Conversion of extracted compound to salt: The dry residue is then dissolved in ethyl acetate (4-5 times the weight of residue) and pH lowered to acidic (3-3.5) by addition of acid, preferably oxalic acid to precipitate Alpha yohimbine (rauwolscine) as salt and recover the same by filtration followed by drying at 60-70 degree C. for 1-2 hours. [0072] 6. Removal of impurities from extract by washing with water and change of pH: The cooled and dried extract of alpha yohimbine (rauwolscine) is purified further by dissolving tn water (5 times tie dry weight) followed by addition of alkali to raise pH to 9-9.5 to precipitate tie compound and recover the same by filtration followed by drying at 60-70 degree C. for 1-2 hours. [0073] 7. Final purification step: The partially purified alpha yohimbine (rauwolscine) compound is dissolved in polar organic solvent such as ethanol, methanol, acetone, ethyl acetate etc. (10 times the weight of residue). The solution is treated with charcoal to remove colour impurities. The clear solution obtained is acidified to pH 2.0-2.5 using acid, preferably hydrochloric acid and cooled to 15-20 degree C. to give maximum precipitation or yield of highly pure (>90%) alpha yohimbine hydrochloride which is filtered aid dried to obtain the final product in form of white/off-white fine powder of Alpha Yohimbine Hydrochloride

[0074] The following example is of die best-contemplated mode of carrying out the invention. The description is made for the purpose of illustrating the general principles of the invention and should not be taken in a limiting sense.

EXAMPLE

[0075] Collection of raw material and powdering: 1000 g leaves of Rauwolfia canescens were collected, dried in the sun and powdered. Extraction was then carried as described in the steps below: [0076] 1. Organic solvantAlkaline Extraction Step: The powder was then mixed with water immiscible solvent viz. any non polar organic solvent such as toluene etc. preferably toluene at 40-50 C. in ratio of 1:4 i.e. 1 part solid and 4 parts solvent and made alkaline by addition of 20-25% ammonia solution, pH 9-9.5 (alkaline) and kept for 4 hours in reactor with stirring followed by filtration. The filtrates/extracts were then transferred to another vessel. The remaining solid material was extracted twice with same solvent as above and the filtrates were pooled in the same vessel. [0077] 2. Organic solvent-Acidic extraction step: The pooled fractions were then made acidic by addition of tartaric acid solution (pH 3.50.5) and extracted with purified water in optimal ratio of 12:1 v/v (volume of pooled fraction: volume of water). As a result the impurities were left behind in the organic layer and the compound of interest was transferred to the acidified aqueous layer. This step was a so performed twice, resulting in high extraction efficiency. [0078] 3. Alkalization of aqueous layer: The aqueous layer containing compound of interest was then basified with 20-25% ammonia solution to pH 9.0-9.5 and extracted with chlorinated solvent such as methylene dichioride, chloroform etc. preferably chloroform, (4:1 v/v; volume of aqueous pooled fraction, volume of solvent) due to which compound of interest was extracted into the organic layer and impurities remained behind in the basic aqueous layer. [0079] 4. Recovery of alpha yohimbine from organic solvent by evaporation of solvent: Alpha yohimbine (rauwolscine) was obtained as dry residue from the organic layer by distillation to evaporate the solvent. [0080] 5. Conversion of extracted compound to salt: To the dry residue remaining behind, ethyl acetate was added (4-5 times the weight of residue) to dissolve the material containing compound of interest completely Then pH was again lowered to acidic by addition of acid, preferably oxalic acid, due to which the desired compound was converted to salt form and precipitated out. The precipitate was collected by filtration using filter cloth and precipitate was dried by heating to 60-70 degree C. for 1-2 hours. [0081] 6. Removal of impurities from extract by washing with water and change of pH: The dried extract was cooled to room temperature and dissolved in water. The compound was precipitated out by making the solution alkaline (pH 9-9.5) by addition or 20-25% ammonia solution while the impurities were left behind in water. Precipitate was recovered by filtration and dried as before. [0082] 7. Final purification step: Dried material was dissolved in polar organic solvent such as ethanol, methanol, acetone, ethyl acetate etc. The solution was treated with charcoal to remove colour impurities. The clear solution obtained was acidified with HCl to pH 2.0-2.5 and cooled to 15-20 degree C. to give maximum precipitation or yield of Alpha Yohimbine Hydrochloride. The precipitate was filtered and dried to obtain the final product in form of white/off-white fine powder of Alpha Yohimbine hydrochloride.

[0083] The yield of the product obtained when using roots and leaves is given in Table 3 below.

TABLE-US-00003 TABLE 3 Yield of alpha yohimbine from roots and leaves of Rauwolfia canesces Assay of S. Herb Batch Yield alpha yohimbine No. part Quantity Yield % (HPLC) 1 Root 500 kg 87 g 0.0174% 82% 2 Leaves 500 kg 2-2.5 kg 0.4%-0.5% 90-93%

[0084] NoveltyThe novelty of the present invention lies in disclosing a simple, commercially viable process for extraction of alpha yohimbine from Rauwolfia species, in high quantities and with high purity (>90%), without use of column chromatography.

[0085] Inventive stepThe technical advancement of knowledge lies in disclosing a commercially viable process for the extraction of alpha yohimbine from Rauwolfia species with the help of organic solvents and alternate steps of acidification and alkalization that provides greater yields and also much higher purity of the compound (>90%) as compared to other processes. The method also excludes the need of expensive and time consuming techniques such as column chromatography. The process has economic importance because it considerably reduces the cost of alpha yohimbine owing to higher yields, faster processing time and simplicity of the process.

INDUSTRIAL APPLICATION

[0086] Alpha yohimbine is used as an aphrodisiac, for impotence, erectile dysfunction, athletic performance, weight lots, exhaustion, angina, hypertension, diabetic neuropathy, and postural hypotension. Due to ts varied application in the medicinal field the extraction process of alpha yohimbine has considerable industrial applications.