Title of Invention	A process for Nymphayol[17-(hexan-2yl)-10,13-dimethylhexadecahydro-1H-cyclopenta[a]phenanthren-3-ol] from Nymphaea stellata with antidiabetic property
Abstract	Nymphayol [17-(hexan-2-yl)-10, 13-dimethylhexadecahydro-lH-cyclopenta[a]phenanthren -3-ol], a novel crystal was isolated from chloroform extract of Nymphaea stellata Willd. Flower. It elicited significant hypoglycemic activity (60.33%) in Streptozotocin-diabetic rats and altered the pattern of glucose tolerance in diabetic rats. In addition, Nymphayol treatment increased plasma insulin levels significantly in streptozotocin-diabetic rats. The hypoglycemic effect of the Nymphayol was comparable with glibenclamide.

Title of Invention

A process for Nymphayol[17-(hexan-2yl)-10,13-dimethylhexadecahydro-1H-cyclopenta[a]phenanthren-3-ol] from Nymphaea stellata with antidiabetic property

Abstract

Nymphayol [17-(hexan-2-yl)-10, 13-dimethylhexadecahydro-lH-cyclopenta[a]phenanthren -3-ol], a novel crystal was isolated from chloroform extract of Nymphaea stellata Willd. Flower. It elicited significant hypoglycemic activity (60.33%) in Streptozotocin-diabetic rats and altered the pattern of glucose tolerance in diabetic rats. In addition, Nymphayol treatment increased plasma insulin levels significantly in streptozotocin-diabetic rats. The hypoglycemic effect of the Nymphayol was comparable with glibenclamide.

Full Text	FIELD OF INVENTION This invention relates to a process for preparation of a novel crystal Nymphayol [ 17-(hexan-2-yl)~ 10,13-dimethylhexadecahydro- l//-cyclopenta[#]phenanthren-3-ol] from Nymphaea stellata Willd. (Nymphaeaceae) flower with antidiabetic property. PRIOR ART Diabetes currently is a major health problem for the people of the world. Diabetes is a chronic disorder of carbohydrate, fat and protein metabolism characterized by elevation of both fasting and post-pradinal blood sugar levels. Globally, the number of people suffering from type 2 diabetes mellitus increased from 30 million to 135 million between 1985 and 1995, and will reach 300 million by the year 2025. The main concern and cost, both human and financial, in relation to diabetes is associated with its long term micro and macro-vascular complications. Diabetes mellitus brings a huge burden of chronic, debilitating disease to our society. Once established, complications can be treated to delay progression, but treatment generally cannot restore normal function or tissue integrity and often the progress of complications is persistent. Preventing or at least delaying the underlying pathological changes that lead to the disease and complications is the goal. Many oral hypoglycemic agents, such as biguanides and sulfonylurea are available along with insulin for the treatment of diabetes mellitus, but these synthetic agents can produce serious side effects, and in addition, they are not suitable for use during pregnancy. In addition, the cost of administering modern antidiabetic drugs is beyond the reach of people in the low-income group and those living in the rural areas. Therefore, search for safe and more effective agents has continued to be an important area of active research. Plants have provided mankind with herbal remedies for many infectious diseases for many centuries and even today, they continue to play a major role in primary healthcare as therapeutic remedies in developing countries. In India herbal medicines have been the bases of treatment and cure for various diseases in traditional methods practiced such as ayurveda, unani and sidha. Many indigenous drugs have been used by practitioners of Ayurvedic system for the treatment of diabetes mellitus in India. The WHO has also recommended the evaluation of the effectiveness of medicinal plants in condition where we lack safe modern drugs. A hypoglycemic action from some treatments has been confirmed in animal models and non-insulin-dependent diabetic patients, and various hypoglycemic compounds have been identified. A botanical substitute for insulin seems unlikely, but traditional treatments may provide valuable clues for the development of new oral hypoglycemic agents and simple dietary adjuncts. A wide array of plant derived active principles representing numerous chemical compounds has demonstrated activity consistent with their possible use in the treatment of diabetes mellitus. Many kinds of natural products, such as alkaloids, glycosides, polysaccharides, peptidoglycans, flavonoids, tannins, steroids, glycopeptides, terpenoids and inorganic ions have been identified. The introduction of these indigenous herbal compounds in the management of diabetes mellitus will greatly simplify the management and make it less expensive. Many plant-derived phytomedicines such as Charantin, a steroidal saponin, obtained from Momordica charantia is known to have an insulin-like activity. Another flavonoid molecule, (-)-epicatechin, has been reported to possess insulin-like activity. Berberine, obtained from Tinospora cordifolia, reduces blood sugar, suggesting that the antihyperglycamic activity of berberine is at least partly due to its ability to inhibit alpha-glucosidase and decrease glucose transport through the intestinal epithelium. We have isolated antidiabetic compound from the plant Nymphaea stellata Willd. flower which has been shown to possess antidiabetic activity in Streptozotocin (STZ) induced-diabetic rats. OBJECTIVE OF THE INVENTION The primary object of the invention is to propose a process for preparation of a novel crystal Nymphayol [ 17-(hexan-2-yl)-10, 13-dimethylhexadecahydro- \H-cyclopenta[a] phenanthren-3-ol] from Nymphaea stellata Willd. flower with antidiabetic property. Another object of this invention is to propose a process for preparation of novel crystal with antidiabetic property to overcome the disadvantages of the prior art. Further object of this invention is to propose a process for preparation of novel crystal with antidiabetic property, which exhibits antidiabetic activity. Still further object of this invention is to propose a process for preparation of a novel crystal with antidiabetic activity, which is not harmful to human beings. STATEMENT OF THE INVENTION According to this invention there is provided a process for preparation of novel crystal Nymphayol [ 17-(hexan-2-yl)-10, 13-dimethylhexadecahydro- 1H-cyclopenta[a]phenanthren-3-ol] from Nymphaea stellata Willd. flower with antidiabetic property comprising steps of: - a) Nymphaea stellata flower powder (1kg) was soaked in n-hexane, extracted and discarded. b) The remaining residue was further extracted with chloroform. c) The chloroform extract was filtered and concentrated at 40°C by using vacuum rotary evaporator and crude extract was obtained. d) Chloroform crude extract was chromatographed on a silica gel column (Merck 70-230 mesh, 400 gm, 3.5 id. x 60 cm) and successively eluted with stepwise gradient of Hexane, Hexane: Chloroform and Hexane: Ethyl acetate system. e) 74 fractions (each 150ml) were collected; each fraction was spotted on a precoated Silica gel 60 F254, 0.25mm thick TLC plate (Merck) and eluted in Hexane: Ethyl acetate (4:1) and fractions with similar Rf values in TLC pattern were pooled together. f) Finally 16 major fractions were obtained. The crystal was obtained from fraction 12. g) The crystal was assayed for the plasma glucose reduction level in Streptozotocin (STZ) induced diabetic rats. h) The crystal possessed strong antidiabetic activity. i) The crystal was studied using crystallography [Bruker axs (Kappa apex2)] and the structure was identified and confirmed. j) Further according to this invention a new crystal was provided with antidiabetic property, which was shown as Nymphayol [ 17-(hexan-2-yl)-10, 13-dimethylhexadecahydro-l//-cyclopenta[a]phenanthren-3-ol]. DETAILED DESCRIPTION OF THE INVENTION WITH REFERENCE TO ACCOMPANYING DRAWINGS k) Fig. 1 shows a flow chart for the isolation of a novel crystal Nymphayol [17-(hexan-2-yl)-10, 13-dimethylhexadecahydro-l//-cyclopenta[a]phenanthren-3-ol] From Nymphaea stellata flowers. NYMPHAEA STELLATA FLOWER POWDER (1 KG) EXTRACT WITH N-HEXANE (DISCARDED) RESIDUE I FURTHER EXTRACT WITH CHLOROFORM I CONCENTRATED USING VACCUM ROTARY EVAPORATOR AT 40°C. CRUDE EXTRACT OBTAINED CHLOROFORM EXTRACT (CRUDE) I SILICA GEL COLUMN CHROMATOGRAPHY (100-200 MESH) ELUTION OF COMPOUNDS WITH INCREASING POLARITY USING HEXANE, CHLOROFORM AND ETHYL ACETATE BASED ON THE TLC PROFOLES, 16 FRACTIONS WERE OBTAINED FRACTION 12 FORMED A CRYSTAL According to figure 1, one kg of Nymphaea stellata flower powder was soaked in 3 liter n-hexane for a period of 48 hour. After extraction the content was discarded. The remaining residue of the plant material was further extracted with 3 liter of chloroform and the extract was concentrated at 40°C using vacuum rotary evaporator. The crude chloroform extract (30g) was adsorbed on to silica gel (90g: 60-120 mesh) and chromatographed on a silica gel (450g 100-200 mesh) column eluted with continuous Hexane, Hexane: chloroform and Hexane : Ethyl acetate gradients. The eluted fractions were evaluated by TLC and combined to give 16 major fractions. Fraction - 1 Hexane (100%) Fraction-2 Hexane: Chloroform (90: 10) Fraction - 3 Hexane: Chloroform (90:10) Fraction-4 Hexane: Chloroform (80:20) Fraction - 5 Hexane: Chloroform (70:30) Fraction - 6 Hexane: Chloroform (50:50) Fraction - 7 Hexane: Chloroform (50:50) Fraction - 8 Hexane: Ethyl acetate (90:10) Fraction - 9 Hexane: Ethyl acetate (90:10) Fraction-10 Hexane: Ethyl acetate (80:20) Fraction -11 Hexane: Ethyl acetate (75:25) Fraction -12 Hexane: Ethyl acetate (75:25) Fraction-13 Hexane: Ethyl acetate (70:30) Fraction -14 Hexane: Ethyl acetate (60:40) Fraction -15 Hexane: Ethyl acetate (50:50) Fraction -16 Ethyl acetate (100%) Crystal was found only in the fraction 12. The crystal is useful for medical and pharmaceutical applications. The following details are found after subjecting the crystal to X- ray diffraction crystallography analysis. Table 1. Crystal data and structure refinement for novel compound, Empirical formula C50 H86 03 Formula weight 735.19 Temperature 293(2) K Wavelength 0.71073 A Crystal system, space group Monoclinic, P21 Unit cell dimensions a = 9.618(5) A alpha = 90.000(5) deg. b = 7.518(5) A beta = 94.483(5) deg. c = 37.491(5) A gamma = 90.000(5) deg. Volume 2703(2) AA3 Z, Calculated density 2, 0.903 Mg/mA3 Absorption coefficient 0.054 mmM F(000) 820 Crystal size 0.3 x 0.1 x 0.1 mm Theta range for data collection 2.12 to 20.45 deg. Limiting indices -9 Reflections collected / unique 16593 / 5337 [R(int) = 0.0406] Completeness to theta = 20.45 99.2 % Absorption correction Semi-empirical from equivalents Max. andmin. transmission 0.9560 and 0.9140 Refinement method Full-matrix least-squares on FA2 Data / restraints / parameters 5337 / 11 / 484 Goodness-of-fit on FA2 1.105 Final R indices [I>2sigma(I)] Rl = 0.0842, wR2 = 0.2505 R indices (all data) Rl = 0.0991, wR2 = 0.2687 Absolute structure parameter -2(4) Extinction coefficient 0.0013(17) Largest diff. peak and hole 0.404 and -0.218 e.AA-3 1.3. Experimental design 1.3.1. Antidiabetic effect of Nymphaea stellata flower chloroform extract (NSFCExt) on normoglycemic and streptozotocin induced hyperglycemic rats. The animals were randomly divided into 5 groups of six animals each as given below. The NSFCExt and glibenclamide were administered orally for 30 days daily using vehicle solution (Dimethylsulfoxide [DMSO] 0.5%; lml/kg b.wt). Group I: Normal control (DMSO) Group II: Normal + NSFCExt (100 mg/kg b.wt.) Group III: Diabetic control (DMSO) Group IV: Diabetic + NSFCExt (100 mg/kg b.wt.) Group V: Diabetic + Glibenclamide (600\|xg/kg b.wt.) 1.3.2. Screening of Nymphayol [17-(hexan-2-yl)-lG, 13-dimethyIhexadecahydro-l/7- cyclopenta [a]phenanthren-3-ol] in normoglycemic and streptozotocin induced hyperglycemic rats, over a period of 2 h. The animals were randomly divided into 9 groups of six animals each as given below. The Nymphayol and glibenclamide were administered orally using vehicle solution (Dimethylsulfoxide [DMSO] 0.5%; lml/kg bodyweight). Group I: Normal control (DMSO) Group II: Normal + Nymphayol (5 mg/kg b.wt.) Group III: Normal + Nymphayol (10 mg/kg b.wt.) Group IV: Normal + Nymphayol (20 mg/kg b.wt.) Group V: Diabetic control (DMSO) Group VI: Diabetic + Nymphayol (5 mg/kg b.wt.) Group VII: Diabetic + Nymphayol (10 mg/kg b.wt.) Group VIII: Diabetic + Nymphayol (20 mg/kg b.wt.) Group IX: Diabetic + glibenclamide (600(ig/kg b.wt.) 1.3.3, Effect of chronic administration of Nymphayol [17-(hexan-2-yl)-10, 13-dimethylhexadecahydro-li/-cyclopenta [a]phenanthren-3-ol] daily for 30 days on plasma glucose, plasma insulin and body weight in normoglycemic and STZ-induced hyperglycemic rats. The animals were randomly divided into 5 groups of six animals each as given below. The Nymphayol and glibenclamide were dissolved in DMSO (Dimethylsulfoxide [DMSO] 0.5%; lml/kg b.wt.) and administered to the rats orally, once daily using an intragastric tube. Group I: Normal control (DMSO) Group II: Normal + Nymphayol (20 mg/kg b.wt.) Group III: Diabetic control (DMSO) Group III: Diabetic + Nymphayol (20 mg/kg b.wt.) Group VII: Diabetic + glibenclamide (600\|xg/kg b.wt.) 1.4. Determination of the plasma glucose and body weight The following biochemical investigation was carried out in our laboratory. Blood was collected by sinocular puncture and on days initial day, 15 day and 30th day after STZ injection. Fasting plasma glucose was estimated using glucose oxidase-peroxidase method (Trinder, 1969). Body weight changes were recorded periodically. 1.5. Determination of plasma insulin Plasma insulin concentrations were determined by radioimmunoassay kit (Pharmacia, Uppsala, Sweden) with a beta metric counter (Cronex, Dupont, France). 1 rye The kit included human insulin as standard and I-labeled human insulin antibody, which cross-reacts similarly with rat insulin. 1.6. Data and statistical analysis Statistical analysis was performed using SPSS software package, version 6.0. The values were analysed by one way analysis of variance (ANOVA) followed by Duncan's multiple range test (DMRT). All the results were expressed as mean ± SD for six rats in each group. P values ANTIDIABETIC ACTIVITY OF NYMPHAYOL fl7-(HEXAN-2-YLM0, 13-DIMETHYLHEXADECAHYDRO- \H- CYCLOPENTAfalPHENANTHREN-3-OL1. 1. 0. Experimental animals Male Wistar strain rats weighing about 200-250gm bred in the Laboratory of Animal Medicine, Centre for Animal Health Studies, Tamilnadu Veterinary and Animal Sciences University, Madhavaram, Chennai, Tamil Nadu, India were used. All the animals were kept and maintained under laboratory conditions of temperature (22°C±2), humidity (45±5%), and 12 hour day: 12 hour night cycle; and were allowed free access to food (standard pellet diet) and water ad libitum. Studies were carried out in accordance with Indian National Law on Animal Care and Use and Committee for the Purpose of Control and Supervision of Experiments on animals and Institutional Animal Ethical Committee Guidelines. 1.1. Source of chemicals Streptozotocin was purchased from Sigma-Aldrich, St. Louis, USA. All other chemicals were of analytical grade obtained from SD Fine, E. MERCK and HIMEDIA, Mumbai, India. 1.2. Experimental induction of diabetes Diabetes mellitus was induced by single intraperitoneal injection of freshly prepared streptozotocin (STZ) (55 mg kg"1 body weight) in 0.1 M citrate buffer (pH -4.5) in a volume of 1 mL/kg bw. STZ injected animals were given 20% glucose solution for 24 h to prevent initial drug-induced hypoglycemic mortality. Diabetes was developed and stabilized in these STZ treated rats over a period of 7 days. The control animals were treated with citrate buffer (pH - 4.5). After 7 days of streptozotocin administration, fasting plasma glucose levels of each rat were determined. Rats with a fasting plasma glucose range of 220-280mg dL"1 were considered diabetic and included in the study. Blood was collected by sinocular puncture. One Kg of Nymphaea stellata Willd. flower dry powder was soaked in 3 ltr n-hexane and the extract was discarded. The remaining residue of the plant material was further soaked in the aspirator bottle with 3 ltr of chloroform and extract was taken in a similar manner. After the extraction the contents were filtered and concentrated at 40°C using vacuum rotary evaporator. After condensation the crude extract was kept in refrigerator at 4°C. 2. 33.6 g of crude extract was obtained from 1 kg of plant powder. 3. 30g of chloroform extract was subjected to column chromatography and 74 fractions were collected. The collected fractions were combined based on the TLC spot to get 16 major fractions. 4. Fraction 12 formed novel crystal, which was studied using XRD and structure of compound was confirmed as Nymphayol [ 17-(hexan-2-yl)-10, 13-dimethylhexadecahydro- l//-cyclopenta[a]phenanthren-3-ol]. It is to be noted that the present invention is susceptible to modification, changes, and adaptations by those skilled in the art. Such modification, changes adaptations are intended to be within the scope of the present invention, which is further set forth under the following claims. ADVANTAGES OF THIS INVENTION Crude chloroform extract of Nymphaea stellata Willd. flowers exhibits significant antidiabetic activity against streptozotocin induced diabetic male wistar rats. The isolated crystal Nymphayol [17-(hexan-2-yl)-10, 13-dimethylhexadecahydro-l//-cyclopenta[a]phenanthren-3-ol], which is confirmed by XRD crystallographic data is found to be responsible for antidiabetic activity. The Nymphayol [17-(hexan-2-yl)-10, 13-dimethylhexadecahydro- l//-cyclopenta[«] phenanthren-3-ol] enhances the antidiabetic activity. Administration of Nymphayol [17-(hexan-2-yl)-10, 13-dimethylhexadecahydro-l//-cyclopenta[a] phenanthren-3-ol] showed a significant hypoglycemic effect and the maximum effect on blood glucose level was observed, over a period of 2 h with the dose of 20mg/kg bw. Chronic administration of Nymphayol [17-(hexan-2-yl)-10, 13-dimethylhexadecahydro-l//-cyclopenta[a]phenanthren-3-ol] was found to significantly decrease the plasma glucose levels from 15th day; in 30th day the glucose level was brought back to near normal. Oral administration of the compound significantly increased the levels of insulin at the end of experiment. The 20mg/kg bw dose showed maximum control among the three different doses and brought the values to near normal levels. A reduction in body weight was observed in STZ-induced diabetic animals, but when the animals were treated with Nymphayol [ 17-(hexan-2-yl)-10, 13-dimethylhexadecahydro-l#-cyclopenta[a] phenanthren-3-ol], the decrease in body weight was minimized and gradual improvement in body weight was observed. The effect of Nymphayol [17-(hexan-2-yl)-10,13-dimethylhexadecahydro-l//-cyclopenta[(7] phenanthren-3-ol] is comparable with standard antidiabetic drug, glibenclamide. The novel compound isolated from Nymphaea stellata flowers does not show any harmful effect to the rats. WE CLAIM 1. A process for preparation of novel crystal of Nymphayol [17-(hexan-2-yl)-10, 13- dimethylhexadecahydro-lH-cyclopenta[a] phenanthren-3-ol] with antidiabetic activity comprising steps of: - i. Nymphaea stellata flower powder (1 kg) was completely extracted with n-hexane. ii. The remaining residue was further extracted with chloroform and concentrated to obtain crude extract. iii. 30g of chloroform crude extract was subjected to column chromatography; 74 fractions eluted with Hexane, Hexane: Chloroform and Hexane: Ethyl acetate were collected. iv. Collected fractions were combined based on the TLC to get 16 major fractions. v. Fraction 12 produced novel compound of Nymphayol [17-(hexan-2-yl)-10, 13-dimethylhexadecahydro-l//-cyclopenta[a] phenanthren-3-ol]. 2. A process as claimed in claim 1, wherein the ratio between the plant powder and chloroform was 1:3 (WW) and plant powder, ethyl acetate was 1:5 (W/V). 3. A process as claimed in claim 2, wherein the flower powder was soaked first in hexane and then using chloroform for period of 48 to 72 hrs to obtain crude extracts. 4. A process as claimed in claim 3, wherein the step of column chromatography was used to collect 74 fractions and then combined based on the TLC to get 16 major fractions. 5. A process as claimed in claim 4, wherein the crystal is obtained from fraction 12 6. A process for preparing a novel crystal Nymphayol [17-(hexan-2-yl)-10, 13- dimethylhexadecahydro-l//-cyclopenta[a] phenanthren-3-ol] substantially herein described and exemplified.

Full Text

FIELD OF INVENTION
This invention relates to a process for preparation of a novel crystal Nymphayol [ 17-(hexan-2-yl)~ 10,13-dimethylhexadecahydro- l//-cyclopenta[#]phenanthren-3-ol] from Nymphaea stellata Willd. (Nymphaeaceae) flower with antidiabetic property.
PRIOR ART
Diabetes currently is a major health problem for the people of the world. Diabetes is a chronic disorder of carbohydrate, fat and protein metabolism characterized by elevation of both fasting and post-pradinal blood sugar levels. Globally, the number of people suffering from type 2 diabetes mellitus increased from 30 million to 135 million between 1985 and 1995, and will reach 300 million by the year 2025. The main concern and cost, both human and financial, in relation to diabetes is associated with its long term micro and macro-vascular complications. Diabetes mellitus brings a huge burden of chronic, debilitating disease to our society. Once established, complications can be treated to delay progression, but treatment generally cannot restore normal function or tissue integrity and often the progress of complications is persistent. Preventing or at least delaying the underlying pathological changes that lead to the disease and complications is the goal.
Many oral hypoglycemic agents, such as biguanides and sulfonylurea are available along with insulin for the treatment of diabetes mellitus, but these synthetic agents can produce serious side effects, and in addition, they are not suitable for use during pregnancy. In addition, the cost of administering modern antidiabetic drugs is beyond the reach of people in the low-income group and those living in the rural areas. Therefore, search for safe and more effective agents has continued to be an important area of active research.
Plants have provided mankind with herbal remedies for many infectious diseases for many centuries and even today, they continue to play a major role in primary healthcare as therapeutic remedies in developing countries. In India herbal medicines have been the bases of treatment and cure for various diseases in traditional methods practiced such as ayurveda, unani and sidha. Many indigenous drugs have been used by practitioners of Ayurvedic system for the treatment of diabetes mellitus in India. The

WHO has also recommended the evaluation of the effectiveness of medicinal plants in condition where we lack safe modern drugs. A hypoglycemic action from some treatments has been confirmed in animal models and non-insulin-dependent diabetic patients, and various hypoglycemic compounds have been identified. A botanical substitute for insulin seems unlikely, but traditional treatments may provide valuable clues for the development of new oral hypoglycemic agents and simple dietary adjuncts. A wide array of plant derived active principles representing numerous chemical compounds has demonstrated activity consistent with their possible use in the treatment of diabetes mellitus. Many kinds of natural products, such as alkaloids, glycosides, polysaccharides, peptidoglycans, flavonoids, tannins, steroids, glycopeptides, terpenoids and inorganic ions have been identified. The introduction of these indigenous herbal compounds in the management of diabetes mellitus will greatly simplify the management and make it less expensive.
Many plant-derived phytomedicines such as Charantin, a steroidal saponin, obtained from Momordica charantia is known to have an insulin-like activity. Another flavonoid molecule, (-)-epicatechin, has been reported to possess insulin-like activity. Berberine, obtained from Tinospora cordifolia, reduces blood sugar, suggesting that the antihyperglycamic activity of berberine is at least partly due to its ability to inhibit alpha-glucosidase and decrease glucose transport through the intestinal epithelium. We have isolated antidiabetic compound from the plant Nymphaea stellata Willd. flower which has been shown to possess antidiabetic activity in Streptozotocin (STZ) induced-diabetic rats.
OBJECTIVE OF THE INVENTION
The primary object of the invention is to propose a process for preparation of a novel crystal Nymphayol [ 17-(hexan-2-yl)-10, 13-dimethylhexadecahydro- \H-cyclopenta[a] phenanthren-3-ol] from Nymphaea stellata Willd. flower with antidiabetic
property.
Another object of this invention is to propose a process for preparation of novel crystal with antidiabetic property to overcome the disadvantages of the prior art.

Further object of this invention is to propose a process for preparation of novel crystal with antidiabetic property, which exhibits antidiabetic activity.
Still further object of this invention is to propose a process for preparation of a novel crystal with antidiabetic activity, which is not harmful to human beings.
STATEMENT OF THE INVENTION
According to this invention there is provided a process for preparation of novel crystal Nymphayol [ 17-(hexan-2-yl)-10, 13-dimethylhexadecahydro- 1H-cyclopenta[a]phenanthren-3-ol] from Nymphaea stellata Willd. flower with antidiabetic property comprising steps of: -
a) Nymphaea stellata flower powder (1kg) was soaked in n-hexane, extracted and discarded.
b) The remaining residue was further extracted with chloroform.
c) The chloroform extract was filtered and concentrated at 40°C by using vacuum rotary evaporator and crude extract was obtained.
d) Chloroform crude extract was chromatographed on a silica gel column (Merck 70-230 mesh, 400 gm, 3.5 id. x 60 cm) and successively eluted with stepwise gradient of Hexane, Hexane: Chloroform and Hexane: Ethyl acetate system.
e) 74 fractions (each 150ml) were collected; each fraction was spotted on a precoated Silica gel 60 F254, 0.25mm thick TLC plate (Merck) and eluted in Hexane: Ethyl acetate (4:1) and fractions with similar Rf values in TLC pattern were pooled together.
f) Finally 16 major fractions were obtained. The crystal was obtained from fraction 12.
g) The crystal was assayed for the plasma glucose reduction level in Streptozotocin (STZ) induced diabetic rats.
h) The crystal possessed strong antidiabetic activity.
i) The crystal was studied using crystallography [Bruker axs (Kappa apex2)] and the structure was identified and confirmed.
j) Further according to this invention a new crystal was provided with antidiabetic property, which was shown as Nymphayol [ 17-(hexan-2-yl)-10, 13-dimethylhexadecahydro-l//-cyclopenta[a]phenanthren-3-ol].

DETAILED DESCRIPTION OF THE INVENTION WITH REFERENCE TO ACCOMPANYING DRAWINGS
k) Fig. 1 shows a flow chart for the isolation of a novel crystal Nymphayol [17-(hexan-2-yl)-10, 13-dimethylhexadecahydro-l//-cyclopenta[a]phenanthren-3-ol] From Nymphaea stellata flowers.
NYMPHAEA STELLATA FLOWER POWDER (1 KG) EXTRACT WITH N-HEXANE (DISCARDED)
RESIDUE
I
FURTHER EXTRACT WITH CHLOROFORM
I
CONCENTRATED USING VACCUM ROTARY EVAPORATOR AT 40°C.
CRUDE EXTRACT OBTAINED
CHLOROFORM EXTRACT (CRUDE)
I
SILICA GEL COLUMN CHROMATOGRAPHY
(100-200 MESH)
ELUTION OF COMPOUNDS WITH INCREASING POLARITY USING HEXANE,
CHLOROFORM AND ETHYL ACETATE
BASED ON THE TLC PROFOLES, 16 FRACTIONS WERE OBTAINED
FRACTION 12 FORMED A CRYSTAL

According to figure 1, one kg of Nymphaea stellata flower powder was soaked in 3 liter n-hexane for a period of 48 hour. After extraction the content was discarded. The remaining residue of the plant material was further extracted with 3 liter of chloroform and the extract was concentrated at 40°C using vacuum rotary evaporator.
The crude chloroform extract (30g) was adsorbed on to silica gel (90g: 60-120 mesh) and chromatographed on a silica gel (450g 100-200 mesh) column eluted with continuous Hexane, Hexane: chloroform and Hexane : Ethyl acetate gradients. The eluted fractions were evaluated by TLC and combined to give 16 major fractions.
Fraction - 1 Hexane (100%)
Fraction-2 Hexane: Chloroform (90: 10)
Fraction - 3 Hexane: Chloroform (90:10)
Fraction-4 Hexane: Chloroform (80:20)
Fraction - 5 Hexane: Chloroform (70:30)
Fraction - 6 Hexane: Chloroform (50:50)
Fraction - 7 Hexane: Chloroform (50:50)
Fraction - 8 Hexane: Ethyl acetate (90:10)
Fraction - 9 Hexane: Ethyl acetate (90:10)
Fraction-10 Hexane: Ethyl acetate (80:20)
Fraction -11 Hexane: Ethyl acetate (75:25)
Fraction -12 Hexane: Ethyl acetate (75:25)
Fraction-13 Hexane: Ethyl acetate (70:30)
Fraction -14 Hexane: Ethyl acetate (60:40)
Fraction -15 Hexane: Ethyl acetate (50:50)
Fraction -16 Ethyl acetate (100%)
Crystal was found only in the fraction 12. The crystal is useful for medical and pharmaceutical applications.

The following details are found after subjecting the crystal to X- ray diffraction crystallography analysis.
Table 1. Crystal data and structure refinement for novel compound,
Empirical formula C50 H86 03
Formula weight 735.19
Temperature 293(2) K
Wavelength 0.71073 A
Crystal system, space group Monoclinic, P21
Unit cell dimensions a = 9.618(5) A alpha = 90.000(5) deg.
b = 7.518(5) A beta = 94.483(5) deg.
c = 37.491(5) A gamma = 90.000(5) deg.
Volume 2703(2) AA3
Z, Calculated density 2, 0.903 Mg/mA3
Absorption coefficient 0.054 mmM
F(000) 820
Crystal size 0.3 x 0.1 x 0.1 mm
Theta range for data collection 2.12 to 20.45 deg.
Limiting indices -9 Reflections collected / unique 16593 / 5337 [R(int) = 0.0406]
Completeness to theta = 20.45 99.2 %
Absorption correction Semi-empirical from equivalents
Max. andmin. transmission 0.9560 and 0.9140
Refinement method Full-matrix least-squares on FA2
Data / restraints / parameters 5337 / 11 / 484
Goodness-of-fit on FA2 1.105
Final R indices [I>2sigma(I)] Rl = 0.0842, wR2 = 0.2505
R indices (all data) Rl = 0.0991, wR2 = 0.2687
Absolute structure parameter -2(4)
Extinction coefficient 0.0013(17)
Largest diff. peak and hole 0.404 and -0.218 e.AA-3

1.3. Experimental design
1.3.1. Antidiabetic effect of Nymphaea stellata flower chloroform extract
(NSFCExt) on normoglycemic and streptozotocin induced hyperglycemic rats.
The animals were randomly divided into 5 groups of six animals each as given below. The NSFCExt and glibenclamide were administered orally for 30 days daily using vehicle solution (Dimethylsulfoxide [DMSO] 0.5%; lml/kg b.wt).
Group I: Normal control (DMSO)
Group II: Normal + NSFCExt (100 mg/kg b.wt.)
Group III: Diabetic control (DMSO)
Group IV: Diabetic + NSFCExt (100 mg/kg b.wt.)
Group V: Diabetic + Glibenclamide (600|xg/kg b.wt.)
1.3.2. Screening of Nymphayol [17-(hexan-2-yl)-lG, 13-dimethyIhexadecahydro-l/7-
cyclopenta [a]phenanthren-3-ol] in normoglycemic and streptozotocin induced
hyperglycemic rats, over a period of 2 h.
The animals were randomly divided into 9 groups of six animals each as given below. The Nymphayol and glibenclamide were administered orally using vehicle solution (Dimethylsulfoxide [DMSO] 0.5%; lml/kg bodyweight). Group I: Normal control (DMSO) Group II: Normal + Nymphayol (5 mg/kg b.wt.) Group III: Normal + Nymphayol (10 mg/kg b.wt.) Group IV: Normal + Nymphayol (20 mg/kg b.wt.) Group V: Diabetic control (DMSO) Group VI: Diabetic + Nymphayol (5 mg/kg b.wt.) Group VII: Diabetic + Nymphayol (10 mg/kg b.wt.) Group VIII: Diabetic + Nymphayol (20 mg/kg b.wt.) Group IX: Diabetic + glibenclamide (600(ig/kg b.wt.)

1.3.3, Effect of chronic administration of Nymphayol [17-(hexan-2-yl)-10, 13-dimethylhexadecahydro-li/-cyclopenta [a]phenanthren-3-ol] daily for 30 days on plasma glucose, plasma insulin and body weight in normoglycemic and STZ-induced hyperglycemic rats.
The animals were randomly divided into 5 groups of six animals each as given below. The Nymphayol and glibenclamide were dissolved in DMSO (Dimethylsulfoxide [DMSO] 0.5%; lml/kg b.wt.) and administered to the rats orally, once daily using an intragastric tube.
Group I: Normal control (DMSO)
Group II: Normal + Nymphayol (20 mg/kg b.wt.)
Group III: Diabetic control (DMSO)
Group III: Diabetic + Nymphayol (20 mg/kg b.wt.)
Group VII: Diabetic + glibenclamide (600|xg/kg b.wt.)
1.4. Determination of the plasma glucose and body weight
The following biochemical investigation was carried out in our laboratory. Blood was collected by sinocular puncture and on days initial day, 15 day and 30th day after STZ injection. Fasting plasma glucose was estimated using glucose oxidase-peroxidase method (Trinder, 1969). Body weight changes were recorded periodically.
1.5. Determination of plasma insulin
Plasma insulin concentrations were determined by radioimmunoassay kit (Pharmacia, Uppsala, Sweden) with a beta metric counter (Cronex, Dupont, France).
1 rye
The kit included human insulin as standard and I-labeled human insulin antibody, which cross-reacts similarly with rat insulin.
1.6. Data and statistical analysis
Statistical analysis was performed using SPSS software package, version 6.0. The values were analysed by one way analysis of variance (ANOVA) followed by Duncan's multiple range test (DMRT). All the results were expressed as mean ± SD for six rats in each group. P values
ANTIDIABETIC ACTIVITY OF NYMPHAYOL fl7-(HEXAN-2-YLM0, 13-DIMETHYLHEXADECAHYDRO- \H- CYCLOPENTAfalPHENANTHREN-3-OL1.
1. 0. Experimental animals
Male Wistar strain rats weighing about 200-250gm bred in the Laboratory of Animal Medicine, Centre for Animal Health Studies, Tamilnadu Veterinary and Animal Sciences University, Madhavaram, Chennai, Tamil Nadu, India were used. All the animals were kept and maintained under laboratory conditions of temperature (22°C±2), humidity (45±5%), and 12 hour day: 12 hour night cycle; and were allowed free access to food (standard pellet diet) and water ad libitum. Studies were carried out in accordance with Indian National Law on Animal Care and Use and Committee for the Purpose of Control and Supervision of Experiments on animals and Institutional Animal Ethical Committee Guidelines.
1.1. Source of chemicals
Streptozotocin was purchased from Sigma-Aldrich, St. Louis, USA. All other chemicals were of analytical grade obtained from SD Fine, E. MERCK and HIMEDIA, Mumbai, India.
1.2. Experimental induction of diabetes
Diabetes mellitus was induced by single intraperitoneal injection of freshly prepared streptozotocin (STZ) (55 mg kg"1 body weight) in 0.1 M citrate buffer (pH -4.5) in a volume of 1 mL/kg bw. STZ injected animals were given 20% glucose solution for 24 h to prevent initial drug-induced hypoglycemic mortality. Diabetes was developed and stabilized in these STZ treated rats over a period of 7 days. The control animals were treated with citrate buffer (pH - 4.5). After 7 days of streptozotocin administration, fasting plasma glucose levels of each rat were determined. Rats with a fasting plasma glucose range of 220-280mg dL"1 were considered diabetic and included in the study. Blood was collected by sinocular puncture.

One Kg of Nymphaea stellata Willd. flower dry powder was soaked in 3 ltr n-hexane and the extract was discarded. The remaining residue of the plant material was further soaked in the aspirator bottle with 3 ltr of chloroform and extract was taken in a similar manner. After the extraction the contents were filtered and concentrated at 40°C using vacuum rotary evaporator. After condensation the crude extract was kept in refrigerator at 4°C.
2. 33.6 g of crude extract was obtained from 1 kg of plant powder.
3. 30g of chloroform extract was subjected to column chromatography and 74 fractions were collected. The collected fractions were combined based on the TLC spot to get 16 major fractions.
4. Fraction 12 formed novel crystal, which was studied using XRD and structure of compound was confirmed as Nymphayol [ 17-(hexan-2-yl)-10, 13-dimethylhexadecahydro- l//-cyclopenta[a]phenanthren-3-ol].
It is to be noted that the present invention is susceptible to modification, changes, and adaptations by those skilled in the art. Such modification, changes adaptations are intended to be within the scope of the present invention, which is further set forth under the following claims.

ADVANTAGES OF THIS INVENTION
Crude chloroform extract of Nymphaea stellata Willd. flowers exhibits significant antidiabetic activity against streptozotocin induced diabetic male wistar rats. The isolated crystal Nymphayol [17-(hexan-2-yl)-10, 13-dimethylhexadecahydro-l//-cyclopenta[a]phenanthren-3-ol], which is confirmed by XRD crystallographic data is found to be responsible for antidiabetic activity. The Nymphayol [17-(hexan-2-yl)-10, 13-dimethylhexadecahydro- l//-cyclopenta[«] phenanthren-3-ol] enhances the antidiabetic activity.
Administration of Nymphayol [17-(hexan-2-yl)-10, 13-dimethylhexadecahydro-l//-cyclopenta[a] phenanthren-3-ol] showed a significant hypoglycemic effect and the maximum effect on blood glucose level was observed, over a period of 2 h with the dose of 20mg/kg bw. Chronic administration of Nymphayol [17-(hexan-2-yl)-10, 13-dimethylhexadecahydro-l//-cyclopenta[a]phenanthren-3-ol] was found to significantly decrease the plasma glucose levels from 15th day; in 30th day the glucose level was brought back to near normal. Oral administration of the compound significantly increased the levels of insulin at the end of experiment. The 20mg/kg bw dose showed maximum control among the three different doses and brought the values to near normal levels.
A reduction in body weight was observed in STZ-induced diabetic animals, but when the animals were treated with Nymphayol [ 17-(hexan-2-yl)-10, 13-dimethylhexadecahydro-l#-cyclopenta[a] phenanthren-3-ol], the decrease in body weight was minimized and gradual improvement in body weight was observed. The effect of Nymphayol [17-(hexan-2-yl)-10,13-dimethylhexadecahydro-l//-cyclopenta[(7] phenanthren-3-ol] is comparable with standard antidiabetic drug, glibenclamide. The novel compound isolated from Nymphaea stellata flowers does not show any harmful effect to the rats.

WE CLAIM
1. A process for preparation of novel crystal of Nymphayol [17-(hexan-2-yl)-10, 13-
dimethylhexadecahydro-lH-cyclopenta[a] phenanthren-3-ol] with antidiabetic activity
comprising steps of: -
i. Nymphaea stellata flower powder (1 kg) was completely extracted with n-hexane.
ii. The remaining residue was further extracted with chloroform and concentrated to obtain crude extract.
iii. 30g of chloroform crude extract was subjected to column chromatography; 74 fractions eluted with Hexane, Hexane: Chloroform and Hexane: Ethyl acetate were collected.
iv. Collected fractions were combined based on the TLC to get 16 major fractions.
v. Fraction 12 produced novel compound of Nymphayol [17-(hexan-2-yl)-10, 13-dimethylhexadecahydro-l//-cyclopenta[a] phenanthren-3-ol].
2. A process as claimed in claim 1, wherein the ratio between the plant powder and
chloroform was 1:3 (WW) and plant powder, ethyl acetate was 1:5 (W/V).
3. A process as claimed in claim 2, wherein the flower powder was soaked first in
hexane and then using chloroform for period of 48 to 72 hrs to obtain crude extracts.
4. A process as claimed in claim 3, wherein the step of column chromatography was
used to collect 74 fractions and then combined based on the TLC to get 16 major
fractions.
5. A process as claimed in claim 4, wherein the crystal is obtained from fraction 12
6. A process for preparing a novel crystal Nymphayol [17-(hexan-2-yl)-10, 13-
dimethylhexadecahydro-l//-cyclopenta[a] phenanthren-3-ol] substantially herein
described and exemplified.

Documents:

1567-CHE-2007 EXAMINATION REPORT REPLY RECEIVED 28-02-2012.pdf

1567-che-2007-abstract.pdf

1567-che-2007-claims.pdf

1567-che-2007-correspondnece-others.pdf

1567-che-2007-correspondnece-po.pdf

1567-che-2007-description(complete).pdf

1567-che-2007-form 1.pdf

1567-che-2007-form 3.pdf

1567-che-2007-others.pdf

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Patent Number

252169

Indian Patent Application Number

1567/CHE/2007

PG Journal Number

18/2012

Publication Date

04-May-2012

Grant Date

30-Apr-2012

Date of Filing

20-Jul-2007

Name of Patentee

SHRI. SAVARIMUTHU IGNACIMUTHU

Applicant Address

ENTOMOLOGY RESEARCH INSTITUTE, LOYOLA COLLEGE, NUNGAMBAKKAM, CHENNAI-34

Inventors:

#	Inventor's Name	Inventor's Address
1	SAVARIMUTHU IGNACIMUTHU	ENTOMOLOGY RESEARCH INSTITUTE, LOYOLA COLLEGE, NUNGAMBAKKAM, CHENNAI-34
2	PANDURANGAN SUBASH-BABU	ENTOMOLOGY RESEARCH INSTITUTE, LOYOLA COLLEGE, NUNGAMBAKKAM, CHENNAI-34

PCT International Classification Number

A61K 35/78

PCT International Application Number

N/A

PCT International Filing date

PCT Conventions:

#	PCT Application Number	Date of Convention	Priority Country
1			NA