INTRODUCTION:

Diabetes Mellitus is a common disease in all over world. Diabetes Mellitus is a group of metabolic disease characterized by hypoglycemia resulting from defects in Insulin secretion. Different species of medical plants are used in the treatment of Diabetes Mellitus. Fresh water Biota are a rich source of structurally novel and biologically achieve metabolites. In the field of research involving bioactive substance of plant origin, a greater interest has now arisen in algae. So, study of antihyperglycemic activity of Terpenoids from fresh water algae. In India this disorder is on alarming condition as compared to most of the developed countries. Despite advances in understanding of the disorder and the management, the mortality and morbidity due to this disease is increasing .The focus has been shifted to treat the various ailments through plant-derived drugs due to their safety efficacy, cultural acceptability and lesser side effects. It is heterogeneous primary disorder of carbohydrate metabolism with multiple etiological factors,it generally involves absolute or relative insulin deficiency, or insulin resistance or both. Whatever the cause, diabetes ultimately lead to hyperglycemia, which is the landmark of this disease syndrome.NIDDM has also been associated with an increased risk for premature arteriosclerosis due to increase in triglycerides and low density lipoprotein levels. About 70-80% of deaths in diabetic patients are due to vascular disease. An ideal treatment for diabetes would be a drug that not only controls the glycemic level, but also prevents the development of arteriosclerosis and other complications of diabetes.

Different species of medicinal plants are used in the treatment of diabetes mellitus. For diabetes treatment before the discovery of insulin by Banting andBest in 1922 the only option were those based on traditional practices(1). In the field of research involving bioactive substances of plant origin a greater interest has now arisen in algae. The first investigation on antibiotic activity of algae(2). Since algal have been used in traditional medicine for a long time(3), and also some algal subatance have bacteriastatic activity, they have been extensively studied by several researchers(4-10)

Without enough insulin, the cells of the body cannot absorb insufficient glucose from the blood: hence glucose levels increase, which is termed as hyperglycemia. If the glucose level in the blood remains high over a long period of time, this can result in long-term damage to organs, such as the kidneys, liver, eyes, nerves, heart and blood vessels. Complications in some of these organs can lead to death. WHO classification of diabetes introduced in 1980 and revised in 1985 was based on clinical characteristics. The two most common types of diabetes were insulin dependent diabetes mellitus (IDDM) of (type-1) and non-insulin dependent diabetes mellitus(NIDDM) OR (TYPE-2).

The sample studied in the present work were collected samples were analysed qualitatively and chemical analysis and extract terpenoids from fresh water algae. Additionally the phytochemical screening aims at the examination of terpenoids Biomass estimation, Chlorophyll estimation, carotenoids estimation, phosphate estimation and hydrocarbon extraction. After extraction preliminary test of crude extract further purified by column chromatography, thin layer chromatography and HPLC. After that I saw spectral study of crude extract with the help of UV, MASS, IR, NMR and experimental design for anti diabetic activity on Swiss Albino mice. Compound Ent-15 hydroxy labda-8,2-ene can inhibit the hyper glycemic activity in mice. In the present study isolation of terpenoid from fresh water algae material and their hyper glycemic activity was determined. These vegetal species could be effective to prevent or retard the development of Diabetes complications due to metabolic disorder.

EXPERIMENT:

Bhopal, the city of lakes situated at 2316 latitude and 77⁰26^’ E longitude, has possession of two lakes an upper and at lower lake. The samples studied in the present work were collected, between January and June 2009, from the Bhopal lakes these specimens represent ten common species belonging to the three major classes of algae, which are chlorophyceae, and Bacillariophyceae. (Table 1).

Table – 1 Showing the percentage occurrence of fresh water algae in Bhopal lake.

Class	Percentage Occurrence	Genera % species
Bacillarion Phyceae	20.15	Cymbella spp. Navicula spp.
Chlorophyceae	30.17	Chlororella spp.
Cyrophyceae	40.17	Anabaera spp. Oscillatoria Spiruling spp.

Each samples were cleaned-up from epiphytes and some were preserved in formalin (4%), others pressed for biological study and identification purposes. Samples were analysed qualitavively enpolying standard biological techniques. Identifications were made employing Standard books and monographs (11-12).

Extraction, Isolation and Purification of Terpenoids

The collected algal materials were thoroughly washed in tap water and air dried at room temperature under shed. The dried algal materials were powered 40 to 60 mesh in size. The powered material was extracted with n-Hexane, petroleum ether and chloroform successively in soxhlet apparatus obtained crude extract (Table 2and3) evaporated to dryness under low temperature and reduce pressure in vacuum evaporator.

Steam and Hydro distillation

Steam distillation, the most common method of essential oil production, involves the flow of steam into a chamber holding the raw plant material. The steam causes small sacs containing essential oil to burst. The oil is then carried by the steam out of the chamber and into a chilled condenser, where the steam once again becomes water. (Hydro-distillation is a similar process where the plant material is boiled, with the resultant steam being captured and condensed). The oil and water are then separated; the water, referred to as a ‘hydrosol’ can be retained as it will have some of the plant essence. Rose hydrosol, for example, is commonly used for it’s mild anti septic and soothing properties, as will as it’s pleasing floral aroma.

Table –2 Showing Percentage Loss in weight on drying and % of Ash contents of fresh water algal material.

Name of algal material	Weight of fresh water algal material	Weight of plant after drying	Loss in weight on drying	% loss in weight	Ash contents
Algal material	2680 gram	535 gram	2145 gram	81.9%	0.064%

Preliminary Test of crude extract for isolation of Terpenoids

A. Libermann- Burchard Test: Small quantity of extract treated with few drops of acetic anhydride, boil and cool then add conc. H₂SO₄ from the side of the test tube brown ring is formed at the junction two layers. Deep red colour indicates the presence of terpenoids.

B. Salkowski test: Trace amount of crude is treated with few drops of conc. H₂SO₄ yellow colour at lower layer indicates presence of terpenoids.

C. Sulfar test: Add small amount of sulfur powder to the test solution, it sinks at the bottom purification of crude extract. After screening and preliminary test of crude extract further purified by Column Chromatography, Thin Layer Chromatography (13).

Detection of compound by following methods:

1. UV- visible Absorption Spectra: The instrument used for present in is UV-Shimadzu UV 160 Spectrophotometer value ranges from Maxima found at 254 nm, 280nm, 470 nm and 510 nm.

2. IR spectra: An invaluable tool in organic structure determination and verification involves the class of electromagnetic (EM) radiation with frequencies between 4000 and 400 cm^-1 (wave numbers). The category of EM radiation is termed infrared (IR) radiation, and its application to organic chemistry known as IR spectroscopy Radiation in this region can be utilized in organic structure determination by making use of the fact that it is absorbed by inter atomic bonds in organic compounds. Chemical bonds in different environments will absorb varying intensities and at varying frequencies. IR-JASCO FT/IR 5300 is used for analysis of data . IR spectroscopy involves collective absorption information and analyzing it in the form of a spectrum. The frequencies at which there are absorptions of IR radiation (“peaks” or “signals”) can be correlated directly to bonds within the compound in question.

Table –3 Showing IR spectra of Ent-15-hydroxy-labda-8, 2-ene, 13 Ediene- 3-one

S.No.	Wave Number	Functional group
1	3752.8	Free O-H stretching
2	3446	Broad inter molecular Hydrogen bonded O-H stretch
3	2927.3	C-H asymmetric stretch
4	2337.8	C=C stretch
5	1643.7	C O stretch
6	1397.1	C-O-H bending bond
7	1223.3	CH₂ wagging
8	1029.9	C-O stretch
9	770	Out of plane aromatic C-H bending

D. Mass spectroscopy: Mass spectrometers are an analytical tool used for measuring the molecular weight (MW) of a sample. MASS-JMS-102 A Mass Spectrophotometer is used for present investigation. Mass spectrometry is based on slightly different principles to the other spectroscopic methods. Molecular weight determined for compound is 308.

E. High Performance Liquid Chromatography (HPLC): High Performance Liquid Chromatography (HPLC) is an analytical technique for the separation and determination of organic and inorganic solutes in any samples especially biological, pharmaceutical, food, environmental, industrial, etc. In a liquid chromatographic process a liquid permeates through a porous solid stationary phase and elutes the solutes into a flow-through detector. The stationary phase is usually in the form of small-diameter (5-10 mm) uniform particles, packed into a cylindrical column. The typical column is constructed from a rigid material (such as stainless steel or plastic) and is generally 5-30 cm long and the internal diameter is in the range of 1-9 mm.

Table –4 Showing HPLC spectra of Ent-15-hydroxy-labda-8, 2-ene, 13 Ediene- 3-one

pK No.	Time	Area	MK	Conc.
1	8.292	32813		2.8955
2	10.817	15166	V	1.3718
3	11.858	67785	V	6.1311
4	13.45	212954	V	19.2615
5	16.233	55737	V	5.8414
6	17.625	112698	V	18.1934
7	22.888	28618	V	2.5884
8	25.825	92166	V	8.3363
9	31.875	49763	V	4.581
10	34.808	146221	V	13.2255
11	39.3	122439	V	11.0745
12	44.342	62485	V	5.6517
13	50.008	23718	V	2.1452
14	68.05	83261	S	7.5309
15	76.908	570	T	0.0516
Total		1105594		100

F. NMR Spectra of Compound: NMR-JEOL FX-4OO Spectrophotometer used to analyses Proton Nuclear Magnetic Resonance of Fresh water algal material .

¹HNMR spectra- δ 0.89 (3H, s, H-20), δ 0.89 (3H, s, H-20), δ 0.99 (3H, s, Me), δ 1.07 (3H, s, H-19), δ 1.25 (3H, s, H-18), δ 1.96 (3H, s, H-16), δ 2.77 (2H, m, H-2), δ 4.32 (2H, d, J = 9.0 Hz, H-15), δ 5.34 (1H, s, H-17), δ 5.36 (1H, s, H-17), δ 7.26 (1H, t, J=9 Hz, H-14).

Experiment of application of compound

Swiss albino mice weighing 24-35 gm of both sexes were purchased from GMC Bhopal Before and during the experiment, the mice were allowed free access to standard pellet diet and water. After randomization into various groups and before initiation of experiment, the mice were acclimatized to the animal house conditions (14-15) at the Department of Biotechnology Bhoj Mahavidhyalya Bhopal. Prior to each study, the animals were made to fast for 12-14 hours but had free access to water (16). All the animal experiments were conducted under the guide line of Institutional Animal Ethical Committee.

Antidiabetic activity:

The rats were randomly divided into six groups consisting of six rats each.

Group 1 - (normal control) consisted of normal rats that neither received alloxan monohydrate nor any drug.

Group 2- served as positive control (diabetic control).

Group 3- Rats were diabetic and treated with atorvastatin (10 mg/kg;p.o).

Group 4- Rats in were diabetic and treated with hydroxychloroquine (200mg/kg).

Group 5- Animals were diabetic and treated with combination of low dose atorvastatin (5mg/kg; p.o.) and hydroxychloroquine (100mg/kg;p.o),

Group 6- Diabetic rats in were treated with combination of atorvastatin (10mg/kg p.o.) and hydroxychloroquine (200mg/kg; p.o.). The drugs were given once daily for 9 days.

Table-5 : Change in body weight of mice treated by Ent-15-hydroxy-labda-8, 2-ene, 13 Ediene- 3-one

Dose	Dosage	Day 0	Day 1	Day 2	%change1	%change2
Crude	300	31.94 ± 1.00	29.42 ± 1.24	30.38 ± 1.43	-7.89	3.26
Compound-1	500	26.68 ± 0.58	25.00 ± 0.96	25.76 ± 0.85	-6.29	3.04
Normal Control 1ml
(Negative) (Vehicle)		25.00 ± 0.96	25.38 ± 0.99	25.52 ± 0.96	1.52	0.55
Diabetic Control
(Positive)		31.92 ± 1.35	29.66 ± 1.09	28.62 ± 1.33	-6.45	-4.15
Glibenclamide	10	30.74 ± 1.18	28.96 ± 1.27	30.20 ± 0.65	-5.79	4.28

Table-6 : Showing Effect of fresh water algal material compound -I on fasting blood glucose level (mg/dl) in normal control and alloxan-induced diabetes mice.

Groups			Day of Treatment
	0	1	7	14
Normal Control	110.00 ± 11.47	105 .40 ± 10.99	111.40 ± 10.94	109.80 ± 7.49
(Negative Control)
Diabetic Control	123.40 ± 9.29	371.20 ± 37.20	391.80 ± 31.26	405.00 ±40.97
(Positive Control)
Glibenclamide	116.40 ± 3.97	349.40 ± 27.57	285.00 ± 22.49	171.00 ± 18.29
Crude Extract	119.20 ± 4.66	335.60 ± 14.01	290.40 ± 26.56	234.00 ± 16.20
300 mg/kg
Compound -I	115.00 ± 4.42	262.60 ± 22.23	241.20 ± 10.16	187.20 ± 17.92
300 mg/kg

The study design was approved Institutional Animal Ethics Committee. The animals were housed in standard environmental conditions of temperature (21 ± 2·), humidity (55 ±10%) and a 12 h light-dark cycle. Rats were supplied with standard pellet diet and water ad libitum.

Oral glucose tolerance test (OGTT)

After 2 weeks of treatment with the plant extracts, the animals were made to fast for 12- 14 hours. Their body glucose level were measured and glucose solution ( 2g/kg body weight) was administered orally in a volume of 1 ml. Blood samples were collected 30, 60 and 120 minutes after administration of glucose in order to evaluate their blood glucose level ( 17).

Data analysis

Data were statistically evaluated by use of one way ANOVA, followed by post hoc Scheffe’s test using version 13 of SPSS software and Microsoft Office Excel 2003. The values were considered to be significant if p<0.05 was obtained.

RESULT AND DISCUSSION:

Of the several hundred thousand plant species around the world, only a small proportion has so far been investigated both phytochemically and pharmacologically. As a single plant may contain thousands of constituents, the possibilities of making new discoveries become evident. The selection of plant material is thus a crucial factor for the ultimate success of the identification of bioactive plant constituents Further purification of crude extract of fresh water algal material with Silica gel in glass column using solvent system (Table 4 to5).After purification two fractions were obtained, Fr. - I and Fr. – II. In the TLC of active fractions yielded two substances in pure form. Both substances were sent to CDRI, Lucknow for spectral analysis.

Spectral analysis showed Compound (I) had the molecular formula C₂₀H₃₀O₃ by mass analysis (m/z 308).

Melting Point of compound (I) - 117 ⁰C to 118 ⁰C

Name of the compound (I) - Ent-15-hydroxy-labda-8, 2-ene, 13 Ediene- 3-one

IR spectra of compound - 3752 cm ^-1, 3446 cm ^-1, 2927 cm ^-1,

2337 cm ^-1 ,1643 cm ^-1, 1223 cm ^-1, 770 cm ^-1

¹HNMR spectra -δ 0.89 (3H, s, H-20), δ 0.89 (3H, s, H-20), δ 0.99 (3H, s, Me), δ 1.07 (3H, s, H-19), δ 1.25 (3H, s, H-18), δ 1.96 (3H, s, H-16), δ 2.77 (2H, m, H-2), δ 4.32 (2H, d, J = 9.0 Hz, H-15), δ 5.34 (1H, s, H-17), δ 5.36 (1H, s, H-17), δ 7.26 (1H, t, J=9 Hz, H-14).

Application:

Ent-15-hydroxy-labda-8, 2-ene, 13 Ediene- 3-one is tested for its anti diabetic activity in mice . This compound is given in crude form as well as in pure form. It also given with a supplement Glibenclamide .The data of tables 4 and 5 show good results with newly synthesized compound . The animals in each group were fasted for 12-14 hours and then the mean blood glucose level was evaluated after oral administration of glucose (2g/kg body weight).In table -4 body weight data is reported . In table -5 blood sugar remarkable control in blood sugar level is reported . Each result is with a mean of 5 mice %change1 indicates the change between day 0 (before alloxan-induction) and day 1 ( after alloxan- induction).% change2 indicates the change between day 1 and day 14. Values are given as mean ± standard deviation for groups of five animals. Values are statistically significant at p<0.05.All these data is reported in table 5 and 6.

CONCLUSION:

In the present investigation a new compound is synthesized and reported from fresh green algae named- Ent-15-hydroxy-labda-8, 2-ene, 13 Ediene- 3-one is among many new bioactive drugs isolated from plants having hyperglycemic effects showed antidiabetic activity equal and sometimes even more potent than known oral hyperglycemic agents Anti diabetic activity of this compound is remarkable in mice .

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Received on 17.11.2013 Modified on 15.12.2013

Asian J. Research Chem. 7(2): February 2014; Page 144-147