Title of Invention

IMPROVED PROCESS FOR THE PREPARATION OF DRUG FROM THE SEEDS OF CAESALPINIA BONDUCELLA FOR THE TREATMENT OF TYPE 2 DIABETES MELLITUS

Abstract

An improved process for the preparation of drugs from the seeds of Caesalpinia species for the treatment of type 2 diabetes mellitus comprising of the following steps: (i) Grinding to moderately coarse powder (all the particles pass through No. 710 sieve and more than 40% through No.250 sieve, i.e., 710/250) of sun dried seed shell of Caesalpinia bonducella F. by gravimetric technique. (ii) Cold Percolation of the plant material obtained from step (i) with solvents for not less than 20 hours. (iii) Centrifuging the materials obtained from step (ii) as herein described. (iv)Drying the extracts by conventional process and finally lyophilized at predetermined temperature as herein described to obtain the dried powder for the treatment of said disease.

Full Text

The present invention relates to an improved process for the preparation of drugs from the seeds of Caesalpinia species for the treatment of type 2 diabetes mellitus. Diabetes mellitus is a serious clinical problem throughout the world, which represents a heterogeneous group of disorders. Some of which can be characterised in terms of specific etiology, and/or pathogenesis. It is a syndrome characterised by chronic hyperglycaemia and disturbance of carbohydrate, fat and protein metabolism associated with absolute or relative deficiencies in insulin action. The fully expressed diabetes is characterised by fasting hyperglycaemia with excessive thirst, polyuria, pruritis, otherwise unexplained weight loss and one or more of the complications associated with or attributable to the disease (Bennett, P. H., Diabetes - Definition and Pathogenesis In: Joslin's Diabetes, Lea and Febiger, 1994, ed. 1.3, Philadelphia, pp. 193 -200). According to World Health Organisation (W.H.O.), diabetes mellitus has been classified under a number of groups. Clinically it can be classified into four major groups as type-I or insulin dependent diabetes mellitus (IDDM), type-II or non-insulin dependent diabetes mellitus (NIDDM), malnutrition related diabetes mellitus (MRDM) and other types of diabetes associated with certain conditions and syndromes like pancreatic disease, disease of hormonal etiology, etc. (Diabetes mellitus, Report of WHO study group, Tech. Rep. Ser., 1985, 727: 1- 113). Among these four types of diabetes mellitus, type-II or NIDDM constitutes about 85% or more of all cases of diabetes. Most forms of NIDDM are associated with a positive family history of disease, associated with obesity (Bennett, P. H., Diabetes - Definition and Pathogenesis In: Joslin's Diabetes, Lea and Febiger, 1994, ed. 13, Philadelphia, pp. 193 -200). There are different modern drugs like biguanides, glibenclamide, etc. which are used for the treatment of diabetes mellitus, specifically of type-II variety. However, most of these drugs have various untoward effects like, lactic acedosis, hypoglycaemic shock, etc. (Katzung, B.G., Pancreatic hormones and anti-diabetic drugs In: Basic and Clinical Pharmacology, 6th ed., Prentice-Hall International Inc., USA, 1995, 637-654). Plant drugs are being searched for their hypoglycaemic and/or anti-diabetic activities for a long time throughout the world. Some of the plant drugs from Stephania hernandifolia, Costus speciosus, Nephrolepsis tuberosa (Mosihuzzaman, M., et al., Hypoglycaemic effects of three plants from eastern Himalayan belt, Diabetes research, 26, 1994, 127-138), Trigonella foenum graecum (Ali.L., et al, Characterisation of the hypoglycaemic effects of Trigonella foenum graecum, Planta medica, 61, 1995, 297-392),etc. have been reported in different literatures for their promising anti-diabetic activities However, most of these plant drugs failed to produce any good marketable anti-diabetic medicine. This failure may be attributed to the incorrect pharmacognosy of the medicinal plant, following up incomplete extraction procedure(s), or use of insensitive and/or inadequate animal models (Chandrasekar, et al, 1988, Blood sugar lowering effect of Tricosanthes dioca Roxb. in experimental rat models, Int. J. Crude-Drug Res., 26: 102-106). Caesalpinia bonducella F. (family Leguminoseae), commonly known as Nala Karanja in Bengali, is a plant used by the tribal people of Andaman and Nicober islands for the relief of the symptoms of diabetes mellitus. The common practice for this purpose 2 is to prepare an aqueous decoction of the seed simply by rubbing on stone with water (Dutta, A. K., Forest Officer, Andaman & Nicober islands, personal communication). This ethnic preparation needed improvement and standardization for its scientific evaluation as anti-diabetic drug and practical viability of cost effective production in large scale. There are some scientific reports (Rao, V.V. et ah, 1994, Hypoglycaemic effect of C. bonducella in rabbits, Fitoterapia, 65, 245-247) for the evaluation of its anti-diabetic properties but screening was not made utilizing ethnological leads. A pilot study was carried out in animal model for hypoglycaemic activity of this plant on the basis of the ethnological reports and significant (p Besides, hypoglycaemic activity the plant has got other biological activities like anti-asthmatic (Gayaraja, S., et ah, 1978, Anti-asthmatic properties of C. honduc leaves, Ind. J. Pharmacol., 10, 86), anti-amoebic and anti-estrogenic (Raghunathan, K. and Mitra, R., 1982, Karanja In : Pharmacognosy of Indigenous Drug, part 1, ed. Raghunathan, K. and Mitra, R., CCRAS, New Delhi, India), anti-anaphylactic and anti-diarrhoeal (Iyenger, M.A. and Pendse, G.S., 1965, Anti-diarrhoeal activity of the nut of Caesalpinia bonducella Flem., Ind. J. Pharm, 27, 307-308), anti-pyretic, anti-diuretic, anthelmintic and anti-bacterial (Neogi, N.C. and Nayak, K.P., 1958, Biological investigation of Caesalpinia bonducella Flem., Ind. J. Pharm., 20 : 95 - 100). Chemically, the seed of Caesalpinia bonducella contains several bitter constituents like acaesalpinia, bcaesalpinia E- caesalpinia, a-terpene.,.caesalpin (1), bonducellin (I), a homoisoflavonoid, caesalpin F- furanoditerpene (Sharina, S.R., et al., Hypoglycaemic. anti-hyperglycaemic and hypolipidemic activities of Caesalpinia bondxicella seeds in rats, J. Ethnopharmacol, 1997 58 39 - 44). However, none of these chemical constituents are tested for any biological properties. In Ayurveda, the Indian traditional system of medicine, the plant is reported to be used in skin disease, leprosy, rheumatoid arthritis, intestinal worms, common cough and cold, fever, hemorrhoids, ulcer, ascites, splenomegaly, bronchial asthma, abdominal colic, etc. (Raghunathan, K. and Mitra, R., 1982, Karanja In: Pharmacognosy of Indigenous Drug, part I, ed. Raghunathan, K. and Mitra, R., CCRAS, New Delhi, India). However, anti-diabetic or hypoglycaemic property of this plant has not been mentioned in any authentic Ayurvedic text. The object of the present invention is the development of an improved process for the preparation of drug for the treatment of type 2 diabetes mellitus (NIDDM) from the seeds of Caesalpinia species. The purpose of present invention is aimed on the basis of the ethnological leads of tribal people of Andaman and Nicober islands, for the selection of the seed shell of Caesalpinia bonducella and preparation is followed accordingly. Aqueous solution is 3 prepared from the moderately coarse powder (all the particles pass through No. 710 sieve and more than 40% through No.25() sieve, i.e., 710/250) of sun dried seed shell of Caesalpinia bonducella F. by gravimetric technique on the basis of ethnological idea. In brief, 40 - 60 g of the powder is dissolved in 90-110 ml of water for 10-14 hours (overnight) and then filtered. The marc left is weighed 40-50 g. The final concentration of the filtrate is, therefore, 40-60 mg/ml. This is used as the test drug for the initial experiments. Primarily the filtrate is tested for acute toxicity study (LD50) including C.N.S. activity and dose response study. The acute toxicity study (LD50) is performed with the doses varying between 1- 5 g / kg b.w. in oral route on swjss albino mice. No lethality or C.N.S. toxicities are observed with these doses. Considering the highest study dose for LD50 (5 g/kg b.w.), dose response study is done below the level of its l/10 value. Responses of different doses like 100, 200, 250, 300 and 400 mg/kg b.w. with the filtrate is observed both on streptozotocin and alloxan induced diabetic models in adult C.F. rats. The doses range between 200 - 300 mg/kg b.w. are found to be significant for lowering of blood sugar levels both in streptozotocin and alloxan induced diabetic models when observed in single dose at the time of ,0, 1, 3 and 5 hours interval (table 1). The filtrate (aqueous solution of Caesalpinia bonducella seed shell) is administered at a dose of 200 - 300 mg/kg b.w./oral route in fasted, fed, glucose loaded (1.5 - 2.5 g/kg b.w./oral), streptozotocin (60 - 70 mg/kg b.w./i.v. in citrate buffer with pH adjusted at 4.5) and alloxan induced (40 mg/kg b.wVi.v.) with respect to vehicle control and glibenclamide (1.5 - 2.5 mg/kg b.wVoral). Blood sugar levels are estimated at 0, 1,3 and 5 hours interval by glucose oxidase-peroxidase method. Significant blood sugar lowering activity (p These results prompted the investigators to undertake detailed and specific study for anti-diabetic properties of two varieties of extracts of Caesalpinia bonducella seeds viz. aqueous extract and 80% ethanolic extracts. The yields of both varieties of extracts are very much remarkable (8 - 16 g /100 g of powder). These extracts are tested both in type-I (IDDM) and type-II (NIDDM) diabetic models in Long Evans rats. Type-I diabetes is produced in adult animals (160-200 g) by injecting streptozotocin (65 mg/kg b.w. /i.p. in citrate buffer with pH adjusting at 4.5). The type-II diabetes, on the other hand, is produced by injecting streptozotocin (90 mg/kg b.w. /i.p. in citrate buffer with pH adjusting at 4.5) to 48 ± 2 hours old Long Evans rats. These rats are selected for test after 3-4 months (90 - 120 days) when the body weight is about 150 - 200 g. The blood sugar levels at pre-dose checking schedule between 9-12 mmol/L is considered for type-II and 20 mmol/L or above for type-I diabetic models. The test compound as well as standard drugs (glibenclamide and metformin) and vehicle control are administered for 5 days with 9 doses in type-II model and 7 days with 7 doses in type-I model. Effects of both aqueous and ethanolic extracts are observed both in these models on the basis of the change of body weight, serum glucose (mmol/L), serum triglycerides (mg/dl) and liver 4 glycogen (mg/g) levels. It appears from the study that both these extracts prevent the fall of body weight (table 3). The serum glucose levels significantly lowered (p In the pathogenesis of hypoglycaemic state of diabetes mellitus, glycogen synthesis in liver and triglycerides levels in blood have a definite relationship. Physiologically glucose is converted into glycogen and stored in liver, muscles, etc. by means of glycogenesis and it is released in the blood as glucose whenever it is needed (Das, D., Carbohydrate metabolism In: Biochemistry, 8th ed., Academic Publishers, Calcutta, 1995, pp 300 - 310). A novel oral hypoglycaemic drug may have property of increasing synthesis of glycogen from glucose in liver. On the other hand, glucose is converted into triglycerides, the major precursor molecule of cholesterol and HDL of blood, through di-hydroxyacetonephosphate (DHAP) pathway. Therefore, hypertriglyceridemia is a common feature of diabetes mellitus. The oral hypoglycaemic drug, which is able to lower the blood sugar level in one hand and serum triglyceride level on the other hand, may be claimed as ideal anti-diabetic drug. The ethnic preparation involved the rubbing of the seed of C. bonducella on stone with water and use the aqueous extract for management of diabetes. This is not a practical method for standardisation and development of drug. Hence the method is improved for the preparation of the test drug by the procedure mentioned hereafter. The aqueous and 80% ethanol extracts of Caesalpinia bonducella are prepared by the methods described here. In brief, the properly identified seed shell of Caesalpinia bonducella is removed, air dried and moderately coarse (710/250) powder is prepared. Distilled water or 80% ethanol at an amount of 800 ml is added separately to powder (l00g each) of the plant. These are soaked for 20 - 24 hours and thereafter filtered through clean mesh. The filtrates are centrifuged at the rate of 20,000 to 25,000 r.p.m. for 20 - 30 minutes. The supernatant decanted and evaporated in a rota-evaporator at the temperature of 40-60° C for 5-7 hours. Finally, the concentrated supernatants are placed in lyophilizer at - 40° C under vacuum pressure of 0.1 to 0.01 Torr for 18-22 hours. These yielded fresh dried extract both aqueous (8-12g) and alcoholic (9 to 15g), from respective solution. Both these extracts are soluble in water. The extracts are preserved in dried sterile airtight glass bottles. In the present study, it is observed that both the aqueous and 80% ethanolic extracts of Caesalpinia bonducella seed shell potentially lower the serum triglyceride levels (35 - 50 % for both aqueous and ethanolic extract) with respect to control and standard drugs (table 5). The amounts of liver glycogen synthesised in liver by the treatment of these extracts are also noticeable (20-30 % for both aqueous and ethanolic extracts with respect to vehicle control treated group). This indicates that the hypoglycaemic property of Caesalpinia bonducella seed shell is due to increased uptake of glucose as glycogen in the liver. These effects are not found to be potent in type-I 5 model (table 5). However, result of 80 % ethanolic extract is found to be better than aqueous extract. It can be concluded that the 80 % ethanolic extract of Caesalpinia bonducella seed shell has potential hypoglycaemic and hypotriglyceridemic activities in type-II diabetic model (NIDDM) owing to its property of increased glycogenesis. Table 1. Dose response study of the aqueous solution of Caesalpinia bonducella F. seed in alloxan and streptozotocin induced diabetic models in C.F. rats Dosage (mg/kg b.w.) /groups Blood sugar (mg/dl) level (Mean ± SEM) in alloxan induced model Blood sugar (mg/dl) level (Mean ± SEM) in Strepto/otocin induced model Ohr. 1 hr. 3hr. 5hr. 0hr. 1 hr. 3 hr 5 hr 100 (n = 6) 332.80 ±13.30 335.00 ± 12.95 334.40 ±13.66 330.60 ± 14.02 253.20 ± 22.30 296.00 ±26.91 280.00 ± 17.28 241.60 ± 34.29 200 (n = 6) 288.20 ±11.00 281.00 ±12.95 267.00 ± 12.95 261.20 ±9.83 390.00 ±16.17 413.00 ± 14.65 357.60 ± 12.72 345.60 ± 24.25 250 (n = 6) 378.40 ±2.57 381.00 ±4.53 340.60* ±11.54 322.00" ±9.14 424.50 ±.16.33 425.50 ± 11.92 354.50* ±23.53 290.74 ± 12.49 300 (n = 6) 326.00 ±17.21 317.40 ±14.19 302.00 ±13.66 298.20 ± 15.47 406.00 ±23.85 404.00 ±22.27 358.80 ±15.12 343.20 ±15.88 400 (n = 6) 353.60 ± 14.68 360.00 ± 16.98 346.60 ±14.64 333.40 ±14.38 368.50 ±12.88 385.80 ± 14.06 350.60 ± 14.48 340.33 ±13.60 Control (n=6) 398.40 ±6.14 417.00 ±10.40 436.80 ±8.36 444.80 ±6.89 316.00 ± 19.22 327.33 ±13.69 312.33 ± 8.98 321.50 ±6.84 'n' indicates number of animal in each group, * p 6 Table 2. Effect of aqueous solution of Caesalpinia bonducella F.(250 mg/kg b.w.) on blood sugar levels in different diabetic models at single dose study with respect to standard and control groups in C.F. rats Experimental models and treatment groups Blood sugar (mg/dl) levels (Mean ± SEM) Ohr. 1 hr. 3 hr. 5hr. Fasted Test (n = 6) Control (n = 5) 105.80 ±4.76 100.33±9.20 127.00±9.02 128.50±3.78 95.20±4.17 112.00±6.22 80.88±4.90 96.66±6.40 Fed Test (n = 6) Control (n = 6) 146.33±10.43 147.50±7.15 157.83±9.83 196.16±7.13 132.00±9.66 157.16±7.17 122.00±13.47 141.83±8.33 Glucose loaded Test (n = 5) Control (n = 5) 115.80±11.68 111.00±3.93 99.20±6.64 119.00±4.25 80.60±6.94** 114.00±3.88 83.86±4.50**: 110.20±4.26 Streptozotocin Test (n = 6) Control (n = 6) Standard (n = 6) 485.83±38.16 403.00±13.50 406.00± 14.22 421.80±33.14 410.33±12.65 366.50±9.47 373.80±26.97 419.66±12.50 388.80±11.95*** 311.60±28.34" 431.66±13.26 297.16±16.18' Alloxan induced Test (n = 6) Control (n = 6) Standard (n = 6) 492.66114.04 494.60±6.40 491.50+8.61 462.33±7.38* 506.60±4.58 443.80±7.23*** 435.66±20.23** 529.00±5.65 398.80±4.57*** 383 00±20.86 ** 545.50±7 62 345.30±9.74*** 'n' indicates number of animal in each group, *p Table 3 : Effect of aqueous and 80% ethanolic extracts of Caesalpinia bonducella on change of body weight with respect to standard and control drugs in L. E. rats. Treatment groups Type I (IDDM) model (Mean± SEM) Type II (NIDDM) model (Mean± SEM) Day 1 Day 3 Day 5 Day 1 Day 3 Day 5 Aqueous extract (n = 7) 178 ± 11 169 ±09 170 ± 10 181± 33 177 ± 34 174 ±35 Ethanolic extract (n = 7) 175 ± 11 170±10 172 ± 11 180 ± 26 175 ± 27 172 ±28 Glibenclamide (n = 7) 174 ± 15 169 ±15 165 ± 14 190 ± 18 185 ± 25 121 ±21 Metformin (n = 7) 165 ± 17 163 ±14 164 ± 13 198 ± 26 180 ± 26 185 ± 26 Vehicle control (n = 7) 165 ± 12 162±11 163 ± 12 175 ± 13 164 ± 13 157± 14 'n' indicates number of animals used in each treatment group 7 Table 4. Effect Of aqueous and ethanolic extracts of Caesalpinia bonducella F. in type-I (IDDM) and type-H (NIDDM) models on L. E. rats with respect to standard and control treated groups Treatment groups Serum glucose (mmol/L) in type-I model (Mean ± SEM) Serum glucose (mmol/L) in type-II (Mean ± SEM) nodel Day 1 Day 7 Day 1 Day 3 Day 5 Control (n=7) 24.73 ± 2.30 23.73 ± 2.14 7.75 ±0.86 7.11 ±0.82 7.44 ± 0.91 Aqueous extract (n = 7) 26.90 ± 3.14 22.34 ± 3.25 7.23 ± 0.59 8.97 ±2.19 5.51 ± 0.72*x Ethanolic extract (n = 7) 25.15 ±4.18 20.22 ± 3.36 8.39 ±1.08 6.93 ± 1.84 5.22 ± 1.72 * x,y Glibenclamide (n=7) 26.83 ± 2.68 18.98 ± 4.93 9.09 ± 0.77 7.44 ± 1 02 6.37 ± 1.86*x Metformin (n = 7) 26.65 + 1.72 14.11 ± 2.07* 8.52 ± 0.85 8,58 ± 1.46 6 10 ± 1 68*y 'n' indicates number of animal in each group, * p Table 5. Effect of aqueous and 80% ethanolic extracts of Caesalpinia bonducella F. on serum triglycerides levels (mg/dl)in type-I and type-II diabetic models on L. E. rats with respect to standard and control treated groups Treatment groups Triglycerides (mg/dl) in type-I (Mean ± SEM) model Triglycerides(mg/dl) in type-II model (Mean ± SEM) Day 1 Day 7 Day 1 Day 5 Control (n=7) 63.47 ± 7.56 61.92 ± 7. 18 60.44 ± 2.47 67.19 ±2.49 Aqueous extract (n = 7) 72.15 ± 11.99 73.87 ± 11 .74 96.49 ± 3.27 74.09 ± 2.87* Ethanolic extract (n = 7) 68.38 ± 6.76 63.13 ± 6. 77 78.88 ± 1.28 58.64 ±0.91* Glibenclamide (n=7) 71.84± 11.05 71.75 ± 10 .87 85.81 ±1.01 55.36 ± 1.03* Metformin (n = 7) 72.67 ± 10.85 71.40± 10 .65 88.93 ± 1.06 55.70 ±1.22* 'n' indicates number of animals in each group, *p 8 Table 6. Effect of aqueous and ethanolic extracts of Caesalpinia bonducella F. on liver glycogen levels in type I and II models in L. E. rats Treatment groups Liver glycogen levels (mg/g) in type-I model Liver glycogen levels (mg/g) in type-II model Control (n =7) 3.62 ±0.38 6.79 ± 0.82 Aqueous extract (n = 7) 10.45 ±1.82* I5.17±O.61** Ethanolic extract (n = 7) 18.92 ± 1.91** 14.38 ±0.77** Glibenclamide (n=7) 18.31 ±2.07** 21.04 ±1.79** Metformin (n = 7) 7.27 ±0.37** 11.89 ±0.83** 'n' indicates number of animal in each group, *p Example: The aqueous and 80% ethanol extracts of Caesalpinia bonducella are prepared as follows: The identified seed shell of Caesalpinia bonducella is removed, air dried and moderately coarse (710/250) powder is prepared. Distilled water or 80% ethanol at an amount of 800 ml is added separately to powder (l00g each) of the plant. These are soaked for 20 - 24 hours and thereafter filtered through clean mesh. The filtrates are centrifuged at the rate of 20,000 to 25,000 r.p.m. for 20 - 30 minutes. The supernatant decanted and evaporated in a rota-evaporator at the temperature of 40-60° C for 5-7 hours. Finally, the concentrated supernatants are placed in lyophilizer at - 40° C under vacuum pressure of 0.1 to 0.01 Torr for 18-22 hours. These yielded fresh dried extract both aqueous (8-l2g) and alcoholic (9 to 15g), from respective solution. Both these extracts are soluble in water. The extracts are preserved in dried sterile airtight glass bottles. 9 We Claim: - 1. An improved process for the preparation of drugs from the seeds of Caesalpinia species for the treatment of type 2 diabetes mellitus comprising of the following steps: i) Grinding to moderately coarse powder (all the particles pass through No. 710 sieve and more than 40% through No.250 sieve, i.e., 710/250) of slin dried seed shell of Caesalpinia bonducella F. by gravimetric technique. ii) Cold Percolation of the plant material obtained from step (i) with solvents for not less than 20 hours. iii) Centrifuging the materials obtained from step (ii) as herein described iv) Drying the extracts by conventional process and finally lyophilized at predetermined temperature as herein described to obtain the dried powder for the treatment of said disease. 10 2. The process as claimed in claim 1, wherein the solvents are water and 80% ethanol. 3. The process as claimed in the preceding claims substantially as herein described.

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