Process for THR preparation of natural crystallized thymol from <i>Monarda citriodora </i>(Jammu Monarda) oil

Abstract

The present invention provides an efficient process for the isolation of natural crystallized thymol product derived from crude natural Monarda citriodora (Jammu Monarda) oil useful for incorporation into foods and beverages in amounts due to strong antimicrobial, antibacterial, antioxidant properties as well as for use as a preservative. The recovery of crystallized thymol product obtained is more than 72% from Hexane-Jammu Monarda oil in a ratio 1:1 under specific conditions. A method for preparing natural thymol in crystal form involves providing crude Jammu Monarda oil in a Flask and gradually reducing the temperature of the flask containing Monarda oil:Hexane in a step-wise manner, thereby producing highly pure crystals (95.5%) within 48 h. The methods disclosed herein are suitable for pharmaceutical GMP.

Claims

1. A process for the preparation of natural crystallized thymol from Monarda citriodora (Jammu Monarda) oil, wherein the steps comprising: [a] filtering the crude Jammu Monarda oil followed by refrigeration at temperature ranging from 0 to 8 C. for 6 to 48 hours; [b] separating the organic layer from the aqueous layer in the refrigerated oil of step and optionally drying the aqueous layer over an anhydrous salt; [c] adding hexane to the organic layer obtained in step [b] followed by refrigeration at a temperature ranging ranging from 0 to 8 C. for 6 to 48 hours; [d] filtering the refrigerated mix of step [c] to separate the thymol crystals from the dethymolised oil.

2. The process as claimed in claim 1, wherein the ratio of hexane to the organic layer is 1:1.

3. The process as claimed in claim 1, wherein refrigeration in step [c] is done at 0 C. for 48 hours.

4. The process as claimed in claim 1, wherein the yield of thymol is in the range of 13 to 71% with 99% purity.

5. The process as claimed in claim 1, wherein the anhydrous salt is selected from sodium sulphate and magnesium sulphate.

Description

BRIEF DESCRIPTION OF DRAWINGS

(1) FIG. 1 Flow chart diagram showing steps for purifying thymol

(2) FIG. 2 Flow chart diagram showing steps for purifying thymol by using Hexane

(3) FIG. 3 Structure of thymol and its various analogues

DEFINITIONS

(4) As used herein, crude thymol oil refers to oils distilled from a Monarda citriodora (Jammu Monarda) plant (belonging to family Lamiaceae), whose oils contain at least 52-85% thymol calculated by GCMS experiment.

(5) As used herein, dethymolised oil (DTO) refers to components of crude thymol oil remaining after at least partially removing thymol from the oil.

DETAILED DESCRIPTION OF THE INVENTION

(6) The plant Jammu Monarda (Monarda citriodora) used for the purposes of the present invention was cultivated at the experimental farm of CSIR-IIIM Chatha, J&K-180009, India which is situated just 11 km far away from Jammu city. The seeds of the crop was sown in the nursery beds in the month of first fortnight of October, 2016 and transplanted the seedling in the field of CSIR-IIIM experimental farm, Chatha in the month of November, 2016. The crop was allowed to grow up to month of April, 2016. Periodic sampling for the essential oil was done during the crop growth season with the help of Clevenger apparatus and accordingly these essential Oil samples were analyzed. For Large scale experiments, the oil was extracted at the experimental field, Chatha with the help of hydro steam distillation unit in 3 hours and 30 minutes. Oil recovery from this crop falls between the ranges from 0.4% to 0.5% depends on the management and time of harvesting of the crop.

(7) The thymol production methods disclosed herein can be used for direct production of highly pure thymol from crude Monarda citriodora (Jammu Monarda) oil. For example, methods disclosed herein can be used to produce pharmaceutical grade thymol. Production of thymol according to various methods disclosed herein may take one week or less. However, in some embodiments, the thymol was obtained within 48 h. In the present invention, the temperature of Monarda oil:hexane mixture (1:1 ratio) has been gradually decreased to yield pure crystals of thymol. Also, the process provides the desired recovery yield of more than 70% thymol from Monarda oil:hexane mixture within 48 hours.

(8) In FIGS. 1 and 2, an exemplary method for thymol production is illustrated. Depending on the embodiment, the method of may include fewer or additional blocks and/to the blocks may be performed in a different order than is illustrated.

(9) Crude thymol oil is provided at block 1. The source and type of the crude thymol oil is Monarda citriodora (Jammu Monarda). In some embodiments, the crude thymol oil contains 1 to 5% water due to crude thymol oil prepared by steam distillation from Monarda citriodora plant material.

(10) The concentration of thymol in the crude Monarda citriodora (Jammu Monarda) oil is not permanent; it remains approximately 52% to 85% thymol by assessed by GCMS analysis and varies according to climate and storage conditions.

(11) The crude thymol oil, in some embodiments, includes few, if any, added organic solvents. By avoiding the use of significant amounts of organic solvent, the methods disclosed herein may not require additional steps of removing organic solvents. Furthermore, the methods disclosed herein may be more economical compared to other methods requiring costly organic solvents. In some embodiments, the crude thymol oil is substantially free of an organic solvent (e.g., not more than trace amounts).

(12) FIGS. 1 and 2 represent purification steps for crude Monarda citriodora (Jammu Monarda) oil performed within the laboratory. Before crystallizing the thymol, seed crystals of thymol may optionally be introduced to aid crystallization. Adding seed crystals of thymol may improve the rate of crystallization.

(13) Seed crystals of thymol may also be introduced, in some embodiments, by rapidly cooling a portion of the crude oil to form seed crystals of thymol. In some embodiments, seed crystals are formed by exposing a portion of the crude Monarda citriodora oil to one or more cooling surfaces having a temperature of not more than about 10 C. For example, experiments were done with various cooling temperature, wherein for instance, the refrigerator having temperature ranging from 0 to 8 C. can be used for making seed crystals.

(14) In some embodiments, after the seed crystals have been formed, the temperature of the one or more cooling surfaces is adjusted to a crystallization starting temperature between 0-8 C. In some embodiments, seed crystals are not introduced, and crystallization occurs at 0-8 C. within 48-72 h without introducing seed crystals of thymol.

(15) The crude thymol oil is crystallized according to FIG. 1 to purify the material. As an example, the crude Monarda citriodora (Jammu Monarda) oil is kept in refrigerator for separation of water layer which is soluble in oil (1-5% water), separated by separating funnel after which the crude oil is dried by anhydrous salt (such as anhydrous sodium sulphate, magnesium sulphate etc.) followed by filtering off the oil, storing in a flask and gradually cooling to obtain crystalline thymol. Yield of obtained crystalline thymol varies according to the cooling temperature (refer Table 1) especially in the absence of seed crystals (preferably a cooling temperature in the range of about 0 C. to 10 C. (because the melting point of thymol crystals is about 48 to 51 C. and boiling point 232 C.). In general, thymol crystal formation may occur over a period of about 48 h.

(16) TABLE-US-00001 TABLE 1 Effect of Different temperatures on the preparation of thymol crystals Exp. Temp. Time Yield No. Quantity ( C.) (h) (%) Observations 1 1 L 8-10 48 No crystal formation was observed till 48 h 2 1 L 6 48 13% Crystal formation was observed after 6 h, and isolated with 95% purity after 48 h. 3 1 L 4 48 38% Crystals were isolated with 99% purity 4. 1 L 0 48 63% Crystals were isolated as white powder with 98-99% purity 5. 1 L 0 48 73% Improvement in yield was observed while addition of Hexane in Oil (1:1) ratio 6 1 L Base/Acid 48 59% Brownish color treatment crystals were isolated (20% NaOH) with 90% purity.

(17) Also The effect of different time duration was observed on thymol crystal formation and 48 h was set as the standardized time.

(18) TABLE-US-00002 TABLE 2 depicts the results of thymol crystal formation when different ratios of hexane to organic layer are used. SI Volume of Volume of Crystal formation No. hexane organic layer (Yield in %)/remarks 1. 250 mL 1 L 63% 2. 500 mL 1 L 65% 3. 1 L 1 L 72% 4. 1.5 L 1 L 70% Note: Slightly decrease in yield was observed when volume of hexane increase from 1 L to 1.5 L.

(19) The initial crystallization temperature (at the start time of the crystallization process, may be a temperature at which the thymol is a liquid. Depending on the embodiment, the initial crystallization temperature was observed to be ranging from 8 to 10 C. (Table 1, experiment 1), In general, during gradual cooling for crystallization of thymol, the crystallization chamber is initially cooled at 0-10 C. for initial 48 hours. The rate of cooling may be determined based on several factors. For example, the concentration of thymol in the crude Jammu Monarda oil may be used in determining the rate of cooling.

(20) In another embodiment, the crude Monarda citriodora (Jammu Monarda) oil after treating as per the process according to FIG. 2, was kept for 48 h at 8 to 10 C., but no crystal formation was found within 48 h at this temperature (Table 1).

(21) In still another embodiment, the cooling temperature was set at 6 C. and crystal formation was observed after 24 h overall 13% pure thymol crystal was obtained within 48 h, Yield may vary in response to time at this set temp.

(22) In yet another embodiment, the initial cooling temperature was set at 4 C. and 0 C. respectively. Yield of thymol increased by decreasing the temperature within 48 hours (Table 1).

(23) In another embodiment, the ratio of organic layer and added hexane is in the range of 1:0.25 to 1:1.5.

(24) In still another embodiment, after treatment of Monarda citriodora (Jammu Monarda) according to FIG. 2, organic layer and added hexane is in a ratio of 1:1, and the initial crystallization temperature was set to 0 C., for 48 h. The obtained yield of thymol was observed to be more than 70%. In this example, using the hexane solvent in a particular ratio increased the crystallized thymol percentage. As described in Table 1, percentage of obtained pure thymol may vary according to cooling periods (more or less than 48 h).

(25) After crystallization, the thymol crystals may optionally be further dried by filtration assembly with a subsequent vacuum. Purity of obtained thymol was monitored by GCMS. In the purification process (during filtration by using vacuum), solid crystal formation was also observed in filtration flask, whereby the vacuum helped in decreasing temperature; which can be a reason to observe the crystals.

(26) The dethymolised oil (DTO) that remains after the crystallization process is stored in different container (because of presence of important molecule such as cervical, cymene etc.). As described above, the dethymolised oil includes the remaining liquid after crystallizing the thymol in the crude Monarda citriodora (Jammu Monarda) oil (e. g., after the crystallization described in FIG. 2)

(27) In some embodiments, cooled water or cooled hexane is passed over the thymol crystals to remove the dethymolised oil from the crystals. Accordingly, passing a cooled solvent over the thymol crystals can improve the purity of the crystals and expedite removing dethymolised oil.

(28) After obtaining purified thymol, it can be transferred to a stock tank. Generally, for transferring the thymol as liquid, the crystallized thymol can be warmed to about 50-60 C. to melt the thymol crystals. The melted thymol can then be transferred to, for example, a stock tank.

(29) In some embodiments, the purified thymol can be formed into particulate for ease of handling. In some embodiments, solid, purified thymol is comminuted (e.g., grinding, milling, cutting, etc.) into particulate. In some embodiments, the particulate are cubic. The cubic/pellet form may be shaped, for example, using a pelletizer, from a thymol melt. Because the melting point of thymol is about 48 C., under cooled mixing conditions, the thymol is changed to a solid state and can further form a desired shape.

(30) The purified thymol can be loaded into the cooling tray mixer that is maintained at a temperature below 10 C. The thymol pellets can be packaged into packing containers for shipping or storage.

(31) Production of Monarda citriodora (Jammu Monarda) Oil

(32) Monarda citriodora crop was cultivated at field CSIR-IIIM, research station Chatha in the year of 2016-17. The crop was transplanted after proper preparation of the land with proper layout and proper crop geometry. All agricultural inputs i.e.; weeding, hoeing, irrigation, fertilizer applications were provided according to the package of practices. The crop was monitored time to time and managed scientifically. It was allowed to mature up to harvesting. Maturity was governed with the help of data recorded for essential oil recovery and quality with the help of Clevenger apparatus. Quality was governed with the help of GCMS. Essential Oil recovery was obtained with the help of periodic samples distilled with Clevenger apparatus. The fresh herbage of Monarda citriodora (5178 kg) was distilled by using hydro steam distillation unit with direct fire system. Essential oil was recovered=20.52 liters. The Essential Oil recovery was 0.40%. 1 L Jammu Monarda oil=760 g Percentage of thymol in Procured Jammu Monarda oil=52%

(33) Note: The concentration of thymol in the crude Monarda citriodora (Jammu Monarda) oil is not permanent; it ranges approximately from 52% to 85% thymol by GCMS analysis and varies according to climate and storage condition.

(34) Thereby, 1 L or 760 g Jammu Monarda oil=395 g (yield 100%) thymol

(35) $\mathrm{Isolation}\mathrm{of}\mathrm{thymol}\text{/}\mathrm{Isolated}\mathrm{thymol}$$\mathrm{crystals}\left(\%\mathrm{yield}\right)=\frac{\mathrm{Actual}\mathrm{yield}}{\mathrm{Theoretical}\mathrm{yield}\left(395g\right)}100$

EXAMPLES

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

Example 1

(37) Crude oil of Monarda citriodora (Jammu Monarda) that contain 52% thymol oil was transferred in 1 Liter glass bottle through a funnel. The crude oil was cooled in the refrigerator for 10-12 hours. After cooling of the oil two layers were formed in the bottle, an organic layer and an aqueous layer (1 to 5% water). The separated oil was stored in a glass bottle and kept in the refrigerator at 8-10 C. for crystallization (table 1). At the end of 48 hours, no crystal formation occurred in the thymol oil.

Example 2

(38) This method illustrates the effect of temperature on crystallization of thymol. Crude oil of Monarda citriodora having 52% thymol was transferred in 1 liter glass bottle and kept in a refrigerator for 10-12 hours. Two layers were formed in the bottle, an organic layer and an aqueous layer. The aqueous layer was separated with the help of separating funnel and the organic layer (oil) was stored in the glass bottle. The bottle was kept in the refrigerator for crystallization and at the end of 48 hours no crystallization was observed. The oil was taken in 1 liter glass bottle and kept in a refrigerator at 6 C. temperature for 48 hours. It was observed that crystallization was initiated after 6 hours in the oil. Dethymolised oil (DTO) was removed from thymol crystals with the help of Buchner funnel and vacuum pump. By this method 50 g (Yield 12.65%) thymol crystal was isolated. The purity of these crystals was monitored by GCMS analysis which was found to be 95.5%.

Example 3

(39) For thymol oil separation from crude oil of Monarda citriodora (52% thymol oil) 1 liter oil was transferred into a glass bottle through a funnel and kept in the refrigerator for cooling. After that, layer formation occurred comprising an organic layer and an aqueous layer. Aqueous layer was separated from the oil and oil was dried over anhydrous salt. Further oil was filtered and transferred to a glass bottle, kept in the refrigerator at 4 C. for 48 hours. At the end of 48 hours, crystal formation was observed in the oil. DTO was separated from thymol crystals using Buchner funnel as well as a vacuum pump and further stored in a bottle. 150 g (Yield 37.97%) thymol crystals were isolated and on GCMS, 99% purity was observed. Therefore, high yield of thymol crystals was obtained by decreasing temperature.

Example 4

(40) Crude oil of Monarda citriodora (52% thymol oil) was transferred in to 1 liter glass bottle through a funnel and kept in a refrigerator for cooling. Due to cooling, layer formation occurred in oil, an organic layer and an aqueous layer. Aqueous layer was separated from oil using separating funnel and anhydrous salt added in the organic layer (oil) for drying. Further, oil was filtered and transferred to a glass bottle, kept in the refrigerator at 0 C. for 48 hours and crystals were observed in the bottle. The crystals were separated from dethymolised oil. From this method, 250 g (yield 63.29% yield) pure white crystals were isolated with 98% purity (according to GCMS analysis). It was observed that in this method at 0 C. an elevated yield of thymol crystals was obtained.

Example 5

(41) Crude oil of Monarda citriodora having 52% thymol was transferred in 1 liter glass bottle and kept in a refrigerator for 10-12 hours. After that two layers were formed in the bottle; an organic layer and an aqueous layer. The aqueous layer was separated with the help of separating funnel and the organic layer (oil) stored in a glass bottle. Then thymol oil with hexane was taken in a glass bottle (1 liter thymol oil and 1 liter hexane), in a ratio of 1:1). The bottle was kept in the refrigerator for crystallization at 0 C. for 48 h. After the stipulated time, crystal formation was noticed in the bottle and later oil was separated from crystals through Buchner funnel and vacuum pump. 285 g (Yield 72.15%) pure thymol crystals were isolated with 95.5% purity.

Example 6

(42) Thymol oil was taken in a glass bottle, first treated with aqueous 20% NaOH solution (500 mL) and kept in a refrigerator for one day. The aqueous layer (Containing thymol salt) and organic layer were separated by separating funnel. The aqueous layer was then transferred to a glass bottle and treated with 20% HCl/water solution (1 L) and stored in a refrigerator again for one day. After one day, brown colored thymol crystals were observed in the water layer and filtered through vacuum pump and Buchner funnel. From this method, 230 g (Yield 58.22%) thymol crystal were observed.

Advantages of the Invention

(43) the developed process allows direct preparation of high purity thymol (approx. 99%) in maximum quantity from crude thymol oil. In the production process, thymol crystals are formed directly from crude oil under controlled cooling of the crude thymol oil in a closed system without the necessity of human contact with the crude Jammu Monarda oil. The methods disclosed herein can be easily adapted to GMP regulations, and may be used to produce pharmaceutical grade thymol.