FORM 2
THE PATENT ACT 1970
&
THE PATENTS RULES, 2003
COMPLETE SPECIFICATION
(See section 10 and rule 13)
1. TITLE OF THE INVENTION
PREPARATION OF TRIACONTANOL AND OCTACOSANOL
2. APPLICANT
(a) NAME: PRAJ INDUSTRIES LIMITED
(b) NATIONALITY: Indian Company
(b) ADDRESS: PRAJ House, Bavdhan
Pune-411021, INDIA
3. PREAMBLE TO THE DESCRIPTION
The following specification describes the invention and the manner in which
it is to be performed.

4. DESCRIPTION
FIELD OF INVENTION
The invention relates to a process for the preparation of a composition of triacontanoi and octacosanol from sorghum DDGS. It particularly relates to the preparation of said compositions useful in preparations of nutraceutical or plant growth enhancer products.
BACKGROUND
Sorghum is a major feedstock used for ethanol production in India. During ethanol production apart from ethanol, DWGS [distiller's wet grain solubles] is produced as a major by-product of grain to ethanol industry. This DWGS is sold as animal feed with a low price. However, unsold DWGS not disposed off a waste residue is a serious problem for ethanol manufacturer. Income generated by selling DWGS is very less compared to overall ethanol plant capacity. DWGS can be dried to distiller's dried grain solubles [DDGS], which can be used as animal feed and can be stored for a longer time period.
Grains contain carbohydrates, proteins, oils and phytochemicals. After fermentation of carbohydrates, other components come to DDGS. Since the starch is used up during fermentation to produce ethanol the concentration of these chemicals in DDGS is approximately three times higher than the grains based on the starch content. DDGS is a rich source of oils, proteins, and phytochemicals. Typically sorghum DDGS contains 7-9 % of oil. Sorghum oil contains good amount of wax in it Also, sorghum oil is rich in many phytochemicals such as policosanols and phytosterols. Policosanols are mixture of long chain alcohols. Two commercially important long chain alcohols present in policosanols are octacosanol (C28H58O) and triacontanoi (C30H62O). Octacosanol is used as a

nutraceutical compound, whereas triacontanol is mainly used as plant growth regulator. Commercially policosanols are recovered from rice bran wax, sugarcane press mud and bees wax. Rice bran and bees waxes are the major source of triacontanol whereas sugarcane press mud is a major source of octacosanol.
The plant growth promoting effects of triacontanol have been well known. Triacontanol is characterized in being effective in very small concentrations as hormones. The mechanism of action is primarily to increase the rate of photosynthesis and thereby to improve the productive capacity of the plants. Triacontanol activates the plant's genes controlling the photosynthesis; these genes in turn produce the enzymes controlling the dark reactions of photosynthesis leading to increase in photosynthetic activities.
Octacosanol helps in improving body's natural ability to process and use oxygen. It is used to treat various disease conditions and as a dietary supplement. It helps the body process oxygen and increase the amount of oxygen that is in the blood and flowing to the muscles. It increases strength and stamina, as well as reaction time. It is used in the treatment of a myriad of diseases, including Parkinson's and amyotrophic lateral sclerosis, or Lou Gehrig's disease. It is also used to treat herpes, skin infections, high cholesterol and hardening of the arteries.
At present no serious attempts are made to recover triacontanol and octacosanol from sorghum DDGS. Current invention gives a method to produce an enriched composition of triacontanol and octacosanol from sorghum DDGS. The main object of the present invention is to develop a process to extract triacontanol and octacosanol from a feedstock like sorghum DDGS.

DESCRIPTION OF DRAWINGS
These and other features, aspects, and advantages of the present invention will become better understood when the following detailed description is read with reference to the accompanying drawings, wherein:
FIGURE 1 depicts a process flow diagram for the preparation of a composition of triacontanol and octacosano) from a sorghum distiller's dry grain solubles (DDGS). Different elements of the process are identified and directional movements of different streams and components formed are shown to describe the features of an embodiment of the present invention.
DETAILED DESCRIPTION OF THE INVENTION
The invention discloses a process for preparation of a composition of triacontanol and octacosanol from sorghum distiller's dry grain solubles (DDGS).
In one embodiment of the invention, the process comprises extracting oil from said soluble by using a first organic solvent; followed by dissolving said oil in a second organic solvent at its boiling point forming a reaction mass; cooling said reaction mass at desired temperature and for desired time period precipitating wax; separating and drying said wax; then reducing aldehyde present in said wax in a third organic solvent using a reducing agent; further washing said reduced wax with water to remove said reducing agent; crystallizing [crystallization - 1] said reduced wax in a fourth organic solvent to form a first solid stream; separating and drying said first solid stream; purifying said first solid stream in a fifth organic solvent by using solid-liquid extraction; and recrystallizing [crystallization -2] said first stream in the fifth organic solvent followed by recovery of a crystallized product. This crystallized product is further crystallized

[crystallization - 3] in a sixth organic solvent to form a final solid stream comprising substantial amounts of triacontanol and octacosanol.
Present invention facilitates efficient recovery of triacontanol and octacosanol from sorghum DDGS by a process comprising six steps namely: 1] extraction of oil from DDGS; 2] dewaxing of said oil to get wax; 3] reduction of said wax; 4] crystallization [crystallization - 1] of said reduced wax; 5] solid-liquid extraction followed by recrystallization [crystallization - 2]; and 6] further crystallization [crystallization - 3]. Each step has one or more elements for performing specific or optional functions as required for achieving selective isolation of triacontanol and octacosanol from sorghum DDGS. A person skilled in the art may appreciate different variations and/ or combinations of these elements that may be used to perform the objects of the invention disclosed herein.
In another embodiment of the invention, a composition of triacontanol and octacosanol is prepared from sorghum DDGS by extracting sorghum oil from sorghum DDGS by using a first organic solvent like hexane. Then the oil is dissolved in a second organic solvent, selected from the group consisting of acetone, hexane, or heptane at its boiling point forming a reaction mass which is further cooled at a temperature of between -3 °C to 18 °C for about 6 h to 16 h leading to precipitation of wax. The wax so formed is separated and dried to remove the solvent. Then wax is dissolved in a third solvent selected from the group consisting of hexane, heptane, ethyl acetate, or xylene, which is further reduced by using sodium borohydride as a reducing agent dissolved in ethanol and water at temperature of between 40 °C to 138 °C for about 0.5 h to 6 h. This step of reduction converts long chain aldehydes to respective alcohols [policosanols]. Then said organic layer is washed with water to remove non-reacted sodium borohydride. Then the reaction mass is subjected to crystallization [crystallization - 1] in the same solvent or in a forth solvent selected from the group consisting of benzene, toluene, cyclohexane,

carbon tetrachloride, isooctane, ethyl acetate, xylene, hexane, or heptane to form a first solid stream which is then separated and dried. The crystallized wax is further purified by using solid-liquid extraction in a fifth organic solvent [polar] like methanol followed by crystallization [crystallization - 2] and solid-liquid separation. Then said crystallized product is further crystallized [crystallization - 3] in a sixth organic solvent like ethyl acetate; then crystals are separated and dried to form final solid stream. First solid stream obtained by this process comprises about 8 % to 30% triacontanol by weight and about 5 % to 15 % octacosanol by weight while final solid stream comprises about 25 % to 55 % triacontanol by weight and about 15 % to 40 % octacosanol by weight.
STEP 1: EXTRACTION OF OIL FROM SORGHUM DDGS
Sorghum oil is extracted from sorghum DDGS by hexane extraction process. Sorghum DDGS contains about 7 - 8 % of oil. By using an organic solvent like hexane for extraction about 90 % of oil is being recovered.
STEP 2: DEWAXING OF SORGHUM OIL
In this step sorghum wax is recovered from said sorghum oil by the process of dewaxing, which involves dissolving the sorghum oi! in solvents like hexane, heptane, or acetone at its boiling point. Then the solvent is cooled gradually to about -3 °C to 18 °C for about 6 h to 16 h. Primary objective of this process is simultaneous extraction of policosanols and aldehydes with wax; i.e. recovering wax enriched with policosanols and long chain aldehydes by the process _ of dewaxing sorghum oil at different temperatures (between -3 °C to 18 °C) and for different time duration (between 6 h to 16 h) by mixing it with an organic solvent in between 1:3 to 1:10 concentration w/w. The wax so formed is separated by centrifugation and then dried in a vacuum oven to remove the solvent.

STEP 3: REDUCTION OF SORGHUM WAX
Wax obtained by dewaxing process as above is rich in policosanols, long chain aldehydes and other components. Aldehydes present in the sorghum wax can be converted to their respective alcohols by a process of reduction using a reducing agent. Wax obtained in said dewaxing process is dissolved in solvent like hexane, heptane, ethyl acetate, or xylene. Then the aldehydes present in the wax are reduced by using an agent like sodium borohydride in solution. The reaction is carried out at about 40 °C to 138 °C for about 0.5 h to 6 h. Sodium borohydride solution is prepared by dissolving sodium borohydride in ethanol and water. Non reacted sodium borohydride is removed by washing with water.
STEP 4: CRYSTALLIZATION OF REDUCED WAX
Reduced wax is crystallized [crystallization - 1] in order to extract policosanois. This crystallization can be carried out in the same solvent that was used for reduction reaction or in the any other solvent like hexane, heptane, ethyl acetate, THF, benzene, toluene, cyclohexane, carbon tetrachloride, isooctane, or xylene. This reaction is carried out at about 5 °C to 30 °C for about 2 h to16 h to get a first solid stream. The first solid stream comprises about 17 % to 30 % triacontanol by weight and about 10 % to 15 % octacosanol by weight.
STEP 5: SOLID-LIQUID EXTRACTION FOLLOWED BY RECRYSTALLIZATION
Then said first solid stream obtained is further purified to get a product enriched with higher content of triacontanol and octacosanol. The first solid stream is subjected to solid-liquid extraction with methanol as a solvent at about 55 °C to 66 °C for about 8 h to 16 h; and allowed to recrystallize [crystallization - 2] in methanol followed by Alteration to obtain an extracted wax [a second solid stream].

STEP 6: FURTHER CRYSTALLIZATION
Then said second solid stream is further crystallized [crystallization - 3] using ethyl acetate at room temperature for about 3 h to 16 h. This results into formation of a final solid stream which comprises about 25 % to 55 % triacontanol by weight and about 15 % to 40 % octacosanol by weight.
The process of current invention has several advantages-over the known methods:
• Present process produces a composition which can be used as a plant growth regulator and a nutraceutical product.
• Simultaneous extraction of policosanols and aldehydes is achieved from sorghum oil i.e. recovering wax enriched with about 15-20 % of policosanols, and about 20 - 35 % of aldehydes by the process of dewaxing of said sorghum oil at relatively higher temperature using an organic solvent at a ratio between 1:3 to 1:10 concentration w/w.
• Further reduction of said aldehydes to alcohols i.e. policosanols, using sodium borohydride as a reducing agent which increases policosanol content of the wax.
• Present invention produces enhanced purity product having a triacontanol content of about 25 - 55 % w/w with octacosanol content of about 15 - 40 % w/w.
• Yet another advantage is use of distillery by-product [DDGSj for production of valuable products like triacontanol and octacosanol.

EXAMPLES
Examples provided below give wider utility of the invention without any limitations as to the variations that may be appreciated by a person skilled in the art. A non-limiting summary of various experimental results is given in the examples and tables, which demonstrate the advantageous and novel aspects of the process for the preparation of a composition of triacontanol and octacosanol from the sorghum grains [DDGS].
EXAMPLE 1
About 25 kg of DDGS was mixed with about 125 litres of hexane and subjected to a solid-liquid extraction method. The mixture was heated up to 60 °C and allowed to reflux for about an hour. Next, said extract was transferred into a distillation unit to distil off hexane. This procedure was repeated three times to afford about 2 kg of sorghum oil containing about 1.1 % of octacosanol and 2 % triacontanol. Extraction efficiency of oil was more than 95%. Next, said 2 kg of sorghum oil was heated with about 6.6 kg of hexane in a jacketed reactor till it dissolved completely. Then the-mixture was cooled slowly to about 15 °C and maintained it at this temperature for about 9 h followed by filtration using a basket centrifuge. Residues containing wax were separated and dried in a vacuum oven at about 60 °C and under 300 mmHg of vacuum for about 5 h. This afforded about 320 gm of wax containing about 6.7 % octacosanol and about 8.8 % of triacontanol. Said filtrate was further subjected to vacuum distillation at about 90 °C and under 10 mmHg of vacuum for about 5 h to remove traces of hexane to obtain de-waxed oil. Further, said 320 gm of wax was then completely dissolved in with about 4.5 kg of hexane at elevated temperature. This was followed by slow addition of a solution of sodium borohydride [about 20 gm of sodium borohydride in about 20 gm of water and about 400 mL of ethanol] to said mixture wit(i refiuxing at about 65 °C

over about 45 min. Then said reaction mixture was maintained at said elevated temperature for about 1 h. Non-reacted sodium borohydride was removed by washing with water. Next, crystallization [crystallization - 1] was allowed to occur in the organic layer at about 22 °C for about 16 h and crystals were separated out by filtration using a basket centrifuge and dried in a vacuum oven for about 5 h. This afforded about 170 gm of final dried wax crystal containing about 18 % of octacosanol and about 28 % of triacontanol. Next, it was subjected to a solid-liquid extraction using methanol in a sohxlet apparatus for about 8 h. Methanol extract was then allowed to recrystallize [crystallization - 2] at room temperature and filtered to obtain about 60 gm of said wax [second solid stream], which was again allowed to further crystallize [crystallization - 3] in ethyl acetate at about 26 °C for about 6 h and filtered using Whatman paper no. 1. These steps afforded about 40 gm of wax containing about 37 % of octacosanol and about 51 % of triacontanol. TABLE 1-1 shows percentage of octacosanol and triacontanol present different fractions.
TABLE 1-1:

Material Amount
(gm) Octacosanol + triacontanol (%w/w) Octacosanol + triacontanol (gm)
Oil 2000 2.90 57.96
Wax 320 15.45 49.53
Reduced Wax [First Solid Stream] 170 45.59 77.50
Final Wax [Final Solid Stream] 40 87.50 34.65
EXAMPLE 2
About 1.6 kg of DDGS was mixed with about 8 litres of hexane and subjected to a solid-liquid extraction method. The mixture was heated up to 60 °C and allowed to reflux for about an hour. Next, said extract was transferred into a distillation unit to distil off hexane. This procedure was

repeated three times to afford about 125 gm of sorghum oil containing about 1.1 % of octacosanot and 2 % triacontanol. Extraction efficiency of oil was more than 95%. Next, said 125 gm of sorghum oil was heated with about 415 gm of hexane in a jacketed reactor till it dissolved completely. Then the mixture was cooled slowly to about 15 °C and maintained it at this temperature for about 9 h followed filtration using Whatman filter paper no.1. Residues containing wax were separated and dried in a vacuum oven at about 60 °C and under 300 mmHg of vacuum for about 5 h. This afforded about 20 gm of wax containing about 6.7 % octacosanol and about 8.8 % of triacontanol. Said filtrate was further subjected to vacuum distillation at about 90 °C and under 10 mmHg of vacuum for about 5 h to remove traces of hexane to obtain de-waxed oil. Further, said 20 gm of wax was then completely dissolved in with about 800 mL of heptane at elevated temperature. This was followed by slow addition of a solution of sodium borohydride [about 1.5 gm of sodium borohydride in about 1.5 gm of water and about 25 mL of ethanol] to said mixture with refluxing at about 95 °C to 98 °C over about 45 min. Then said reaction mixture was maintained at said elevated temperature for about 1 h. Non-reacted sodium borohydride was removed by washing said organic layer with water. Next, crystallization [crystallization- 1] was allowed to occur in said organic layer at about 8 °C for about 16 h and crystals were separated out by filtration using Whatman paper no. 1 and dried in a vacuum oven for about 5 h. This afforded about 9.2 gm of final dried wax crystal containing about 18 % of octacosanol and about 32 % of triacontanol.

EXAMPLE 3
About 0.4 kg of DDGS was mixed with about 2 litres of hexane and subjected to a solid-liquid extraction method. The mixture was heated up to 60 °C and allowed to reflux for about an hour. Next, said extract was transferred into a distillation unit to distil off hexane. This procedure was repeated three times to afford about 32 gm of sorghum oil containing about 1.3 % of octacosanol and 2 % triacontanol. Extraction efficiency of oil was more than 95%. Next, said 32 gm of sorghum oil was heated with about 320 gm of hexane in a jacketed reactor till it dissolved completely. Then the mixture was cooled slowly to about 3 °C and maintained at this temperature for about 9 h followed by filtration using Whatman paper no.1. Residues containing wax were separated and dried in a vacuum oven at about 60 °C and under 300 mmHg of vacuum for about 5 h. This afforded about 5 gm of wax containing about 8.1 % octacosanol and about 10 % of triacontanol. Said filtrate was further subjected to vacuum distillation at about 90 °C and under 10 mmHg of vacuum for about 5 h to remove traces of hexane to obtain de-waxed oil. Further, said 5 gm of wax was then completely dissolved in about 100 mL of ethyl acetate at elevated temperature. This was followed by slow addition of a solution of sodium borohydride [about 0.31 gm of sodium borohydride in about 0.31 gm of water and about 5 mL of ethanol] to said mixture with refiuxing at about 77 °C to 80 °C over about 45 min. Then said reaction mixture was maintained at said elevated temperature for about 1 h. Non-reacted sodium borohydride was removed by washing said organic layer with water. Next, crystallization was allowed to occur in the organic layer at room temperature for about 16 h and crystals were separated out by filtration using Whatman paper no.1 and dried in a vacuum oven for about 5 h. This afforded about 4.5 gm of final dried wax crystal containing about 15 % of octacosanol and about 24 % of triacontanol.

EXAMPLE 4
Sorghum wax was recovered from dewaxing of sorghum oil. The process of dewaxing was carried out using various non-polar solvents and at different temperatures so as to obtain the wax enriched in policosanols and long chain aldehydes. TABLE 4-1 shows recovery of policosanols by dewaxing by using various solvents at different temperatures.
TABLE 4-1:

POLICOSANOLS [PC] RECOVERED IN WAX (RECOVERY = PC IN WAX/PC IN THE INITIAL OIL)

1 Hexane 1 10 16 3 4.78 6.54 73.62
2 Heptane 1 10 16 3 5.77 7.83 79.74
3 Acetone 1 10 16 3 3.53 4.93 79.01
4 Hexane 1 10 16 -3 5.19 7.16 70.44
5 Heptane 1 10 16 -3 5.28 7.25 63.70
6 Acetone 1 10 16 -3 3.19 4.49 79.80
7 Hexane 1 10 16 12 6.42 8.84 74.06
8 Heptane 1 10 16 12 6.04 8.4 64.66
9 Acetone 1 10 16 12 4.58 5.87 74.84
10 Hexane 1 10 16 8 6.13 7.77 75.81
11 Heptane 1 10 16 8 6.33 7.94 71.72
12 Acetone 1 10 16 8 3.85 5.41 72.41
13 Hexane 1 10 16 0 4.54 6.41 76.26
14 Heptane 1 10 16 0 3.35 6.52 41.37
15 Acetone 1 10 16 0 3.62 5.01 72.88

EXAMPLE 5
TABLE 5-1 shows recovery of policosanols after reduction of wax in present of different solvents.
TABLE 5-1:

Solvent Yield of Purity octacosanol + Octacosanol
crystallized wax triacontanol (% w/w) + triacontanol
obtained (% recovery
w/w) on initial wax taken (% w/w)
Ethyl Acetate 67.84 38.52 147.7
Heptane 46 49.7 152.6
Hexane 53.12 45.59 156.47
EXAMPLE 6
To determine the efficacy of triacontanol from said first solid stream, prepared according to the process of this invention, as a plant growth enhancer a solution of about 0.1 % of triacontanol was prepared. About 0.5 gm of 28 % triacontanol from said first solid stream containing product was dissolved in 20 mL of ethanol forming an ethanol solution. Next, in a separate three neck flask 99 mL of water was mixed with 0.2 gm of a non-ionic surfactant like a polysorbate (TWEEN 80) and heated to 80 °C. Then, ethanol solution containing triacontanol was then gradually added to the water solution in such manner that rate of addition should match with the rate of evaporation of ethanol. The traces of the ethanol were removed using vacuum distillation. This solution was cooled and volume was adjusted to 100 mL by addition of extra water. Then this 0.1 % triacontanol solution was used for field trials on the growing tomato crop. Two different concentrations 2 and 5 mL/L [i.e. 10 ppm and 25 ppm of triacontanol] were used to check its efficiency. Solutions were sprayed after 26, 45 and 65 days of transplantation on growing crop. TABLE 6-1 lists the results of trials with respect to control in terms of plant height, number of branches, fruit

setting percentage, average fruit weight, average yield per pickings, and total yield of the tomatoes at the end of the trial. It was observed that there is substantial increase in the yield of tomatoes as compared to control when 2 mL/L and 5 mL/L of solutions were used.
TABLE 6-1

Feature Field No. Triacontanol amount [0.1 % solution in water]
Control 2mL/L 5mL/L
Plant height [cm] 1 121 140 126
2 129 145 130
Number of branches 1 6 9 7
2 7 9 8
Fruit setting (%) 1 66 75 73
2 68 76 74
Average fruit weight
(gm) 1 92 125 112
2 98 130 112
Average yield per 1 281 331 303
pickings (kg) 2 285 335 304
Total number of 1 20 21 21
pickings 2 23 24 24
Total Yield (MT) 1 5.62 6.95 6.36
2 6.56 8.04 7.20
While the invention has been particularly shown and described with reference to embodiments listed in examples, it will be appreciated that several of the above disclosed and other features and functions, or alternatives thereof, may be desirably combined into many other different systems or applications. Also that various presently unforeseen and unanticipated alternatives, modifications, variations, or improvements therein may be subsequently made by those skilled in the art which are also intended to be encompassed by the following claims. Although the invention has been described with reference to specific preferred embodiments, it is not intended to be limited thereto, rather those having

ordinary skill in the art will recognize that variations and modifications may be made therein which are within the spirit of the invention and within the scope of the claims.

5. CLAIMS
WE CLAIM:
1. A process for preparation of a composition of triacontanol and octacosanol comprising:
(a) providing a distiller's dry grain soluble;
(b) extracting oil from said soluble by using a first organic solvent;
(c) dissolving said oil in a second organic solvent at its boiling point forming a reaction mass;
(d) cooling said reaction mass at desired temperature and for desired time period precipitating wax;
(e) separating and drying said wax;
(f) reducing aldehyde present in said wax in a third organic solvent using a reducing agent;
(g) washing said reduced wax with water to remove said reducing agent;
(h) crystallizing said reduced wax in a fourth organic solvent to form a first solid stream;
(i) separating and drying said first solid stream;
(j) purifying said first solid stream using a fifth organic solvent, and recrystallizing it in said fifth solvent to form a second solid stream; and

(k) further crystallizing said second stream in a sixth organic solvent to form a final solid stream.
2. The process of claim 1, wherein said
(a) soluble is obtained from sorghum;
(b) first organic solvent is hexane;
(c) second organic solvent is selected from acetone, hexane, heptane or a combination thereof;
(d) third organic solvent is selected from ethyl acetate, hexane, heptane, xylene or a combination thereof;
(e) reducing agent is sodium borohydride;
(f) forth organic solvent is selected from benzene, toluene, cyclohexane, carbon tetrachloride, isooctane, ethyl acetate, xylene, hexane, heptane or a combination thereof;
(g) cooling is performed at temperature of about -3 °C to 18 °C for about 6 to 16 hours;
(h) reduction is performed at temperature of about 40°C to 138 °C for about 0.5 to 6 hours;
(i) crystallization steps (h), (j) and (k) are performed at temperature of about 5 °C to 30 °C for about 2 to16 hours;
(j) first solid stream is recovered by centrifugation or filtration;
(k) first solid stream is dried in a vacuum oven at about 60 °C for about 4 hours;
(I) fifth organic solvent is methanol; and

(m) said sixth organic solvent is ethyl acetate.
3. The process of claim 1, wherein said first solid stream comprises about 8 % to 30 % triacontanol by weight and about 5 % to 15 % octacosanol by weight.
4. The process of claim 1, wherein said final solid stream comprises about 25 % to 55 % triacontanol by weight and about 15 % to 40 % octacosanol by weight.
5. A composition of triacontanol and octacosanol according to the process of claim 1.
6. A product comprising a composition of triacontanol and octacosanol according to the process of claim 1.