Process for production of anti-diabetic compound in root culture of catharanthus roseus

PROCESS FOR PRODUCTION OF ANTI- DIABETIC COMPOUND IN ROOT CULTURE OF CATHARANTHUS ROSEUS

FIELD OF INVENTION

The present invention relates to a process of generating root culture of Catharanthus roseus for production of anti-diabetic compounds. The present invention also relates to identification, purification and characterization of anti diabetic compound, serpentine.

BACKGROUND

Plants, being static organism, need to cope with the changing environment for its survival, growth and development. During evolution plants have developed systems to produce 'secondary metabolites' which help the organism to function better in the fluctuating environmental conditions. Numerous plant secondary metabolites possess interesting biological activities and find applications as pharmaceuticals, insecticides, dyes, flavors and fragrances. The plant kingdom is a rich source of such biochemicals which are used by the human race for nutrition, healing and recreation. One of these products is alkaloid which constitutes a large group of plant secondary metabolites. Human recognition of alkaloids is as old as civilization, since these substances have been used in drugs, medicines, dyes, teas and poisons for 4000 years.

Catharanthus roseus (L.) G. Don, an apocynaceous plant, has been known for its considerable medicinal value in folklore in various countries for a long period. C. roseus is commonly known as periwinkle or Madagascar periwinkle because this plant is believed to have its centre of origin in Madagascar, from where it has spread world wide. It is grown mainly as an ornamental plant in homes, gardens and parks for its colored flowers and also cultivated as a medicinal plant. The plant has been used for centuries to treat diabetes, high blood pressure, asthma, constipation and menstrual problems. More recently, extracts from Madagascar periwinkle have been shown to be effective in the treatment of various kinds of leukemia, skin cancer, lymph cancer, breast cancer and Hodgkin's disease.

Terpenoid indole alkaloids (TIAs), an important group of secondary metabolites, are produced by C. roseus. It produces more than 130 TIAs and research on TIA biosynthesis is particularly focused on the pharmaceutical applications of several of its final compounds. Ajmalicine and serpentine, produced in roots are used in the treatment of circulatory disorders, whereas, vincristine and vinblastine produced in leaves are powerful anticancer drugs. These TIAs are known to be the end products of a complex process, comprising of the involvement of at least thirty five intermediates, thirty enzymes, thirty biosynthetic genes, eight regulatory genes and seven inter and intracellular compartments.

Despite significant efforts, the production of these compounds in cell suspension culture has been an elusive goal. Since late eighties, hairy roots have been demonstrated to have great potential for production of plant secondary metabolites. Hairy roots are generated when Agrobacterium rhizogenes, a plant pathogenic gram negative soil bacterium, transfers its T-DNA from the root inducing (Ri) plasmid to the host genome. The stable integration of T-DNA in plant cell genome and its expression leads to hairy root syndrome. The advantages of hairy root cultures over cell suspension cultures are their fast growth in hormone free media along with genetic and biochemical stability.

The potential of hairy roots of C. roseus has been investigated previously for the production of root specific TIAs, though, in most cases hairy roots have been generated from horticultural varieties of C. roseus (Bhadra and Shanks 1997).

The growing incidence of insulin resistance and Type2 diabetes is seriously threatening human health globally. At the dawn of this new millennium when WHO has announced type 2 diabetes as an epidemic disease of the world, we are still confronted with the major question i.e. what really causes this insidious disease. Typel diabetes or insulin dependent diabetes mellitus (IDDM) is basically due to autoimmune mediated destruction of pancreatic β islets resulting insulin deficiency. Type2 diabetes or non insulin dependent diabetes mellitus (NIDDM) is characterized by insulin resistance, it is the global epidemic disease as more than 90-95% cases are of this kind, while Type 1 is only about 3-5% (Zimmet et al, 2001).

It is now well established that elevated free fatty acids (FFA) in circulation is associated with the loss of insulin sensitivity causing insulin resistance. Incubation of muscle strips or cultured muscle cells with FFA reduces insulin stimulated glucose uptake (Kopelman et al., 2000) by producing defects in insulin signaling. Although FFA are known to play a key role in promoting the loss of insulin sensitivity causing insulin resistance, but how they oppose insulin activity is not yet clear. Novel PKC isoforms, especially PKCε plays a major role in impairing insulin sensitivity (Ikeda et al., 2001). Over expression and chronic activation of PKCε has recently been implicated in the decrease of insulin receptor (IR) numbers in the skeletal muscle of diabetic sand rat (Moller, 2001). Very recently, we have demonstrated FFA induced phosphorylation of PKCε in 3T3L1 adipocytes and skeletal muscle cells in in vitro culture (Yu et al., 2002). Surprisingly, FFA effected PKCε phosphorylation independent of PDKl (Yu et al., 2002). Translocation of phospho- PKCε (pPKCε) to the nucleus coincided with the inhibition of IR gene expression that consequently impaired insulin signaling in skeletal muscle cells (Yu et al., 2002). However, the most important question still remains unanswered i.e. how FFA induced pPKCε could downregulate the expression of IR gene.

Transcription of IR gene requires the assembly of multiprotein complex that activates IR gene promoter. This multiprotein complex includes a nuclear protein, HMGAl (a High mobility group protein), an architectural transcription factor and two ubiquitously expressed transcription factors, SpI and c/EBPβ. HMGAl induces transcriptional activation of IR gene by recruiting SpI and c/EBPβ to the IR promoter. Mutational interference of HMGAl binding site in the IR promoter abolished its binding and that adversely affects recruitment of SpI and c/EBPβ to the IR promoter and this blunts promoter activation. HMGAl therefore, plays a critical regulatory role for IR gene transcription. Another aspect of insulin resistance and type2 diabetes has also been studied by several authors i.e. the involvement of NF-κB. Several evidences that have accumulated recently indicated that lipid-induced elevation of NF-κB activity may play an important role in insulin resistance and type2 diabetes (Martin et al., 2000). Activation of inhibitor of KB kinase (IKK) phosphorylates IKB, an inhibitor of NF- KB; phosphorylated IKB is degraded releasing NF-κB for its translocation to the nucleus which regulates number of gene expression. Interestingly, a short term lipid infusion in humans induces skeletal muscle insulin resistance along with a reduction of IKB levels, indicating the activation of NF-κB during insulin resistance. Number of reports linked skeletal muscle insulin resistance to the excessive activity of IKB and NF-κB (Griffin et al., 1999; Pessin and Saltiel, 2000; Bhattacharya et al., 2007). FFA induced activation of NF-κB in L6 cell causes insulin resistance, while inhibition of NF-κB activation prevents the insulin resistance of L6 myotubes (Martin et al., 2000). It has been reported that FFA induces the expression of interleukin 6 (IL-6) in human skeletal muscle through the activation of NF-KB. It has also been shown that FFA stimulates IL-6 production in skeletal muscle cells via the mediation of PKC and NF-κB activation where IL-6 produces prodiabetic effects (Joshi et al., 1996). Reports are there that show a link between FFA induced NF-κB activation and novel PKCs in effecting insulin resistance (Dey et al., 2005). We have recently shown that PKCε plays a major role in FFA-induced insulin resistance of skeletal muscle cell and adipocytes as it is involved in the downregulation of insulin receptor gene expression.

Although above mentioned pathways indicate the mechanisms those are involved in causing insulin resistance and Type2 diabetes but there is no report on the interference of these pathways by any inhibitor which may offer a therapeutic choice in controlling this insidious disease. There is practically no drug which addresses Type2 diabetes where insulin sensitivity is remarkably lost (insulin resistance) due to FFA except thiazolidinediones (TZD) classes of drugs such as rosiglitazones, pioglitazones which improves insulin sensitivity. TZD basically decrease FFA from the surroundings of insulin target cells by allowing FFA to be uptaken by adipocytes. This they do by activating PPARγ, the orphan receptor which in turn stimulates glycerol kinase gene expression that results in activation of more glycerol molecules in the adipocytes resulting greater formation of di and tri glycerides along with the facilitated entry of FFA into the adipocytes. Hence TZD classes of drugs do not interfere with the pathways through which FFA causes insulin resistance but they reduce the circulatory FFA level and that improves the situation.

SUMMARY OF THE INVENTION

The present invention is based on the surprising discovery that serpentine is useful for the treatment of diabetes and insulin resistance condition. The present invention discloses that administration of serpentine to a subject reduces blood glucose level significantly.

One aspect of the present invention provides a process of production of serpentine in hairy root culture of Catharanthus roseus, the process comprises transforming a cell, tissue or any part of Catharanthus roseus with Agrobacterium rhizogenes to obtain hairy roots; culturing the hairy roots in a growth medium comprising B5 medium and sucrose for about 20 to 40 days; andisolating the serpentine from the hairy root using conventional method, wherein the hairy roots are capable of producing serpentine in the range of 1.12 to 1.80 mg/g of dry weight of the hairy roots.

Another aspect of the present invention provides a composition for treatment of diabetes and insulin resistance, the composition comprising a therapeutically effective amount of serpentine and a pharmaceutically or nutritionally acceptable carrier.

Another aspect of the present invention provides a composition for treatment of diabetes and insulin resistance, the composition comprising a therapeutically effective amount of serpentine and a pharmaceutically or nutritionally acceptable carrier, wherein the serpentine is obtained by • transforming a cell, tissue or any part of Catharanthus roseus with

Agrobacterium rhizogenes to obtain hairy roots;

• culturing said hairy roots in a growth medium comprising B 5 medium and sucrose for about 20 to 40 days to obtain biomass of hairy root culture; • drying the biomass at room temperature;

• homogenizing the dried biomass to obtain a powder;

• extracting the powder with a solvent to obtain the hairy root extract, and

• isolating the serpentine from the hairy root extract using conventional method. wherein the extract comprises serpentine in the range of 1.12 to 1.80 mg/g dry weight of the hairy roots.

Another aspect of the present invention provides use of serpentine for the preparation of a medicament for the treatment of diabetes and insulin resistance. Yet another aspect of the present invention provides a method of treatment of diabetes and insulin resistance comprising administrating an effective amount of serpentine to a subject in need thereof.

Yet another aspect of the present invention provides a process of making Catharanthus roseus hairy root extract, the process comprises • transforming a cell, tissue or any part of Catharanthus roseus with

Agrobacterium rhizogenes to obtain hairy roots;

• culturing the hairy roots in a growth medium comprising B5 medium and sucrose for about 20 to 40 days to obtain biomass of hairy root culture; • drying the biomass at room temperature;

• homogenizing the dried biomass to obtain a powder; and

• extracting the powder with a solvent to obtain the hairy root extract, wherein the biomass of hairy root culture is capable of producing serpentine in the range of 1.12 to 1.80 mg/g dry weight of the biomass of hairy root culture. Yet another aspect of the present invention provides a composition for treatment of diabetes and insulin resistance, the composition comprising a Catharanthus roseus hairy root extract, wherein the hairy root extract is prepared by

• transforming a cell, tissue or any part of Catharanthus roseus with Agrobacterium rhizogenes to obtain hairy roots; • culturing the hairy roots in a growth medium comprising B5 medium and sucrose for about 20 to 40 days to obtain biomass of hairy root culture;

• drying the biomass at room temperature;

• homogenizing the dried biomass to obtain a powder; and • extracting the powder with a solvent to obtain the hairy root extract, wherein the hairy root extract comprises serpentine in the range of 1.12 to 1.80 mg/g dry weight of the hairy roots.

BRIEF DESCRIPTION OF ACCOMPANYING DRAWINGS

Figure 1 shows the results of the experiment related to the isolation and purification of serpentine and its effects on loss insulin activity due to fatty acid. (Ia) shows the single sharp peak identified as serpentine by 2D-NMR and Mass spectrometry in Catharanthus roseus root extract prepared with methanol and then subjected to Diaion HP-20 chromatography followed by Sephadex LH-20 column chromatography. The active fraction was subsequently run through HPLC Reverse Phase chromatography. (Ib) shows graphical representation of the percentage increase in the glucose uptake of the L6 skeletal muscle cells incubated for 6 hours in the presence or absence (control-C) of palmitate (P) or palmitate plus serpentine followed by 30 min incubation with insulin (I+P+Ser) or insulin (I) alone.

Figure 2 shows the insulin stimulated GFP-Glut4 translocation to the cell membrane from cytosol by administration of serpentine

Figure 3 Insulin stimulated Insulin receptor (IP: IR) (a) tyrosine kinase (p-Ty), (b) phosphorylation of insulin receptor substrate (p-IRSl) and (c) Phosphatidyl Inositol-3 kinase (p-PI3K): these are the signal molecules of insulin action. All of them were inhibited by free fatty acid (FFA) i.e palmitate (P) suggesting a downregulation of insulin signal. Addition of serpentine (Ser) blocks FFA induced inhibition, this permitted the rescue of insulin signaling molecules damage thereby revoking insulin activity.

Figure 4 a, b shows insulin stimulated phosphorylation of PDKl and Akt/PKB inhibited in palmitate incubated L6 skeletal muscle cells Figure 5a) shows detection of palmitate incubation of skeletal muscle cells by RT-PCR for 6h demonstrating decrease in the IR gene transcription.

5b) shows Real-time PCR data showing a highly significant (p < 0.001) quantitative decrease of IR mRNA due to palmitate.

5 c) shows consequent reduction of IRβ protein due to the inhibition of IR mRNA Figure 6 shows the results of the effect of serpentine on genetically modified diabetic mice

Figure 7 shows growth of NCHR- 5 in liquid culture medium. Roots were grown in four different liquid culture medium Sl, S2, S3, S4 and solid ¹A strength of gamborg's B5 basal medium. Growth index was calculated on the basis of dry weight. [GI= Increase in dry weight/Initial dry weight X 100]

DETAILED DESCRIPTION OF THE INVENTION

The present invention relates to a process for production of anti-diabetic compounds in root culture of Catharanthus roseus. Further present invention describes the preparation of the extract from the root culture and isolation of pure molecule from the root culture extract. The invention also describes isolation, purification and characterization of the compound by testing the biological activity at every step of purification. The compound isolated from the root culture extract was found to have anti-diabetic properties. This compound on further analysis was found to be serpentine. The present inventors have discovered that hairy root extracts of Catharanthus roseus is useful for the treatment of diabetes and insulin resistance condition. Further analysis of the extract revealed that the extract comprises serpentine which has the effect in lowering the blood plasma glucose level. This result is surprising and unexpected. The present invention provides an economically valuable use of hairy root culture and extract of the hairy root culture for production of high amount of serpentine, in particular as a pharmaceutical composition for treating the metabolic syndrome and diabetes, more in particular a pharmaceutical composition for type 2 diabetes, and as a source for extracting high amount of serpentine, which has anti-diabetic activity. Furthermore, the production of high amount of serpentine in hairy root culture having anti diabetic activity obtained according to the process of the present invention represents unexpected and surprising result.

One embodiment of the present invention provides a process for production of antidiabetic compound in root culture of Catharanthus roseus, wherein the root culture was obtained by infecting Catharanthus roseus plant, plant cell, or any part thereof with Agrobacterium rhizogenes.

Another embodiment of the present invention provides a growth medium for root culture of Catharanthus roseus plant for high production of anti-diabetic compound.

One embodiment of the present invention provides a process for production of anti- diabetic compound in root culture of Catharanthus, wherein the root culture was obtained by infecting Catharanthus plant, plant cell, or any part thereof with Agrobacterium rhizogenes in a growth medium for high production of anti-diabetic compound.

Another embodiment of the present invention provides use of serpentine for the treatment of metabolic disorders and/or syndromes like diabetes.

Still another embodiment of the present invention provides use of serpentine for the treatment of type2 diabetes.

Yet another embodiment of the present invention provides a method of treatment of type 2 diabetes using a composition comprising the serpentine. In another embodiment of the present invention, serpentine from hairy root of Catharanthus roseus extract is prepared by the technique known in the art.

The present invention further provides a pharmaceutical preparation comprising effective amount of serpentine isolated from the root culture of Catharanthus roseus.

The present invention further provides a pharmaceutical preparation comprising effective amount of serpentine isolated from the root culture of Catharanthus roseus with a pharmaceutically acceptable carrier.

In another embodiment of the invention relates to the selection of carriers. The carrier is selected from nutrients such as proteins, carbohydrates, sugars, talc, magnesium stearate, cellulose, calcium carbonate, starch gelatin paste and pharmaceutically acceptable carriers, excipient, diluent or solvent.

Still another embodiment, the pharmaceutical preparation disclosed in the present invention can be administered orally in the form of powder, granules or capsule.

Still another embodiment, the dried product can be administered orally in the form of powder, granules or capsule. In yet another embodiment, the fraction is administered at a dose level of 20 μg/kg body weight for a period 30 days.

In yet another embodiment, it relates to a. method for treating animals and human beings.

In another embodiment of the present study, plant material is used for extraction with appropriate solvent such as methanol, halogenated solvent in a percolator or the equipment known in the art.

One embodiment of the present invention provides alcohol extraction of genetically modified Catharanthus roseus root. The extracted solution, after evaporation was evaluated for bioactivity and a compound showing biological activity was identified as Serpentine by comparing its physical data as well as its infrared (IR), nuclear magnetic resonance ¹H NMR, ¹³C NMR and mass spectral data with those of as authentic sample. In- vivo activity of the isolated compound ie serpentine was analysed and it was found that it blocks the lipid induced pathways for developing insulin resistance and type2 diabetes. Therefore, this compound has great promise as a therapeutic choice to deal with the type2 diabetes. Another embodiment of the present invention provides treatment of diabetic mice with serpentine. To examine the effect of serpentine in reducing the blood glucose level in diabetic mice, leptin receptor knock out genetically modified mice (GM) was selected. These GM diabetic obese mice have consistent high sugar level (between 400-550 mg/dl). 20 μg/ 100 gm body weight of serpentine was fed along with the normal diet for 15 days. The average glucose level, in the plasma of these GM diabetic mice in the experimental group was 450 mg/dl and it was reduced significantly (p<0.01) due to the administration of serpentine.

In another embodiment of the present invention there is provided effect of hairy root extract on genetically modified diabetic mice. These GM diabetic obese mice have consistent high sugar level (between 400-550 mg/dl). Concentrated hairy root extract comprising approximately 20 μg/ 100 gm body weight of serpentine was fed along with the normal diet for 15 days. After administration of hairy root extract significant reduction (p<0.01) in glucose level in the plasma of these GM diabetic mice in the experimental group was observed. In accordance with the present invention there is provided a process of production of serpentine in hairy root culture of Catharanthus roseus, the process comprises

• transforming a cell, tissue or any part of Catharanthus roseus with Agrobacterium rhizogenes to obtain hairy roots;

• culturing the hairy roots in a growth medium comprising B 5 medium and - sucrose for about 20 to 40 days; and

• isolating the serpentine from the hairy root using conventional method wherein the hairy roots are capable of producing serpentine in the range of 1.12 to 1.80 mg/g of dry weight of the hairy roots.

One embodiment of the present invention provides the process of production of serpentine in hairy root culture of Catharanthus roseus, the process comprises

• transforming a cell, tissue or any part of Catharanthus roseus with Agrohacterium rhizogenes to obtain hairy roots;

• culturing the hairy roots in a liquid growth medium comprising B 5 medium and sucrose for about 20 to 40 day; and

• isolating the serpentine from the hairy root using conventional method wherein the hairy roots are capable of producing serpentine in the range of 1.12 to 1.80 mg/g of dry weight of the hairy roots

Another embodiment of the present invention provides a composition for treatment of diabetes and insulin resistance, the composition comprising a therapeutically effective amount of serpentine and a pharmaceutically or nutritionally acceptable carrier. Another embodiment of the present invention provides a composition for treatment of diabetes and insulin resistance, the composition comprising a therapeutically effective amount of serpentine and a pharmaceutically or nutritionally acceptable carrier, wherein the serpentine is obtained by

• transforming a cell, tissue or any part of Catharanthus roseus with Agrobacterium rhizogenes to obtain hairy roots;

• culturing the hairy roots in a growth medium comprising B 5 medium and sucrose for about 20 to 40 days to obtain biomass of hairy root culture;

• drying the biomass at room temperature; • homogenizing the dried biomass to obtain a powder; • extracting the powder with a solvent to obtain the hairy root extract, and

• isolating the serpentine from the hairy root extract using conventional method. wherein the extract comprises serpentine in the range of 1.12 to 1.80 mg/g dry weight of the hairy roots.

Yet another embodiment of the present invention provides solvent for extraction of the root powder, wherein the solvent is selected from a group consisting of methanol, ethanol, isopropyl alcohol, dichloromethane, chloroform, ethyl acetate, water or a mixture thereof. Yet another embodiment of the present invention provides the composition for treatment of diabetes and insulin resistance, the composition comprising a therapeutically effective amount of serpentine and a pharmaceutically or nutritionally acceptable carrier, wherein said effective amount is 10 μg /100 gm body weight to 50 μg /100 gm body weight of serpentine preferably 20 μg/100 gm of serpentine.

Yet another embodiment of the present invention provides a dosage of the composition comprising serpentine, wherein the dosage comprises 10 μg /100 gm body weight to 50 μg /100 gm body weight of serpentine preferably 20 μg/100 gm of serpentine. Further, the present invention provides use of serpentine for the preparation of a medicament for the treatment of diabetes and insulin resistance.

In another embodiment of the present invention there is provided use of serpentine for the preparation of a medicament for the treatment of diabetes and insulin resistance, wherein diabetes is type 2 diabetes. The present invention also provides a method of treatment of diabetes and insulin resistance comprising administrating an effective amount of serpentine to a subject in need thereof.

In one embodiment of the present invention provides a a method of treatment of diabetes and insulin resistance comprising administrating an effective amount of serpentine to a subject in need thereof, wherein said effective amount is 10 μg /100 gm body weight to 50 μg /100 gm body weight of serpentine preferably 20 μg/100 gm of serpentine.

In another embodiment of the present invention provides a method of treatment of diabetes and insulin resistance comprising administrating an effective amount of serpentine to a subject in need thereof, wherein the diabetes is type 2 diabetes.

Another embodiment of the present invention provides a process of making Catharanthus roseus hairy root extract, the process comprises

• transforming a cell, tissue or any part of Catharanthus roseus with Agrobacterium rhizogenes to obtain hairy roots; • culturing the hairy roots in a growth medium comprising B 5 medium and sucrose for about 20 to 40 days to obtain biomass of hairy root culture;

• drying the biomass at room temperature;

• homogenizing the dried biomass to obtain a powder; and

• extracting the powder with a solvent to obtain the hairy root extract, wherein the biomass of hairy root culture is capable of producing serpentine in the range of 1.12 to 1.80 mg/g dry weight of the biomass of hairy root culture.

Another embodiment of the present invention provides a process of making Catharanthus roseus hairy root extract, the process comprises

• transforming a cell, tissue or any part of Catharanthus roseus with Agrobacterium rhizogenes to obtain hairy roots; • culturing the hairy roots in a liquid growth medium comprising B5 medium and sucrose for about 20 to 40 days to obtain biomass of hairy root culture;

• drying the biomass at room temperature;

• homogenizing the dried biomass to obtain a powder; and

• extracting the powder with a solvent to obtain the hairy root extract, wherein the biomass of hairy root culture is capable of producing serpentine in the range of 1.12 to 1.80 mg/g dry weight of the biomass of hairy root culture.

Yet another embodiment of the present invention provides solvents for extraction of serpentine from the hairy root extract, wherein the solvent is selected from a group consisting of methanol, ethanol, isopropyl alcohol, dichloromethane, chloroform, ethyl acetate, water or a mixture thereof.

Yet another embodiment of the present invention provides a hairy root extract obtained by

• transforming a cell, tissue or any part of Catharanthus roseus with Agrobacterium rhizogenes to obtain hairy roots; • culturing the hairy roots in a growth medium comprising B5 medium and sucrose for about 20 to 40 days to obtain biomass of hairy root culture;

• drying the biomass at room temperature;

• homogenizing the dried biomass to obtain a powder; and

• extracting the powder with a solvent to obtain the hairy root extract, wherein the biomass of hairy root culture is capable of producing serpentine in the range of 1.12 to 1.80 mg/g dry weight of the biomass of hairy root culture.

Further embodiment of the present invention provides use of the hairy root extract for preparation of a medicament for the treatment of diabetes and insulin resistance.

Yet another embodiment of the present invention provides a composition comprising the hairy root extract as disclosed in present invnetion and a pharmaceutically acceptable carrier. Yet another embodiment of the present invention provides a composition for treatment of diabetes and insulin resistance, said composition comprising a Catharanthus roseus hairy root extract, wherein the hairy root extract is prepared by

• transforming a cell, tissue or any part of Catharanthus roseus with Agrobacterium rhizogenes to obtain hairy roots; • culturing the hairy roots in a growth medium comprising B5 medium and sucrose for about 20 to 40 days to obtain biomass of hairy root culture;

• drying the biomass at room temperature;

• homogenizing the dried biomass to obtain a powder; and • extracting the powder with a solvent to obtain the hairy root extract, wherein the hairy root extract comprises serpentine in the range of 1.12 to 1.80 mg/g dry weight of the hairy roots.

The composition as disclosed in the present invention elevated tissue fatty acid content when administered to a subject in need thereof. The composition as disclosed in the present invention reduces abnormally elevated blood glucose levels when administered to a subject in need thereof.

EXAMPLES

It should be understood that the following examples described herein are for illustrative purposes only and that various modifications or changes in light will be suggested to persons skilled in the art and are to be included within the spirit and purview of this application and the scope of the appended claims.

Example 1

Generation of hairy roots

Hairy roots were generated from C. roseus var. Prabal which is known to accumulate high alkaloid content. The explants were taken from 2-4 months old seedling grown under aseptic conditions. The leaf (1x1 cm²) and hypocotyl (~lcm) explants were cut from seedling and preincubated on Gamborg's B 5 medium for 24 h in dark. Explants were infected with a wild type virulent strain of Agrobacterium rhizogenes strain A4. Single colony of A. rhizogenes was inoculated in 50ml of YEB medium and grown overnight at 28⁰C with continuous shaking at 200 rpm until OD₆₀₀ reached 0.5-0.6 and this culture was directly used for transformation. Needle was dipped in Agrobacterium culture and explant was pricked with the same needle on its adaxial surface leaving Agrobacterium cell culture at the wound site of explant. The infected explants were then co-cultivated on half strength of Gamborg's B5 culture medium at 25±2°C for 4 d in dark. To remove excess of Agrobacterium from explant after 4 day of co-cultivation, the explants were transferred to Gamborg's B5 medium containing ampicillin (lOOmg/1) and tetracycline (20mg/l) and incubated in dark till hairy roots generated from or near the site of infection. 1-2 cm long roots were excised and transferred to the same medium. Hairy roots were maintained at 25±2°C, 16/8 h light and dark photoperiod and transferred to fresh medium every 5-6 weeks. A total of 250 individual hairy root clones were derived from C. roseus var Prabal, however only 50 root clones survived and were successfully maintained on 'ΛB5 supplemented with 3% sucrose and pH adjusted to 5.75. These root clones were named as NCHRl -50 (N-NIPGR; C- Catharanthus; HR-Hairy root). Root clones showed enormous variability in growth pattern, type of branching and number of lateral roots. For alkaloid analysis, tissue was dried under shade and dried tissue was homogenized and total alkaloid was extracted. Quantification of alkaloid was done by HPLC analysis.

Fifty independent hairy root clones were analyzed for the accumulation of serpentine in these hairy root clones which ranged from .07-1.4 mg/g dw. In C. roseus, intermediates or TIAs themselves are known to be cytotoxic for the growth of hairy root cultures which leads to the selection of low- or non-producing lines after long term culture. The root clone which accumulated 1.4± 0.17 mg/g dw serpentine showed very slow growth and could not survive. One fast growing root clone NCHR-5 was obtained in this screening process for a high alkaloid yielding hairy root clone which stably produced high amount of serpentine (1.12±0.13 mg/g dw) over its 4 years of maintenance.

Hairy roots are known as solid phase biocatalyst and not dispersed in liquid like suspended cells. For commercial exploitation of hairy roots cultures, growth of root clones should be optimized in liquid culture medium for biomass accumulation. The selected root clone NCHR-5 which accumulates high amount of serpentine was grown in liquid culture conditions. Different concentrations of Gamborg's B5 medium supplemented with different sucrose concentration (Sl-l/4^th B5+3% sucrose, S2-l/4^th B5+1.5% sucrose, S3-l/10^th B5+3% sucrose, S4-l/10^th B5+1.5% sucrose) were used in order to optimize culture conditions for growth of C, roseus hairy root clones in liquid culture for higher biomass accumulation and was compared with the growth on 1/2 B5 solid phase medium. 0.6 to 0.8 gm of fresh weight of hairy root from 4 weeks old cultures was inoculated in 250 ml Erlenmeyer flask containing 40 ml liquid growth medium and was kept on orbital shaker at 80 rpm under 25±2°C and 16/8 h light/dark photoperiod. Similar inoculum was used for the study of root growth on solid phase medium. Roots were harvested after 7 d, 15 d, 21 d and 30 d in triplicate and growth index was calculated on dry weight basis.

Differential growth of NCHR-5 was observed in different culture medium (Fig. 7). No significant difference in growth was found during first week of growth period, which represented the lag phase of growth in suspension medium. After 15 d, root clones showed maximum growth in Sl followed by S2. In case of solid phase medium, the growth of root clone was the minimum till 14 d after which the growth was increased exponentially. After 30 d of culture, root clone showed maximum biomass accumulation in S2 and Sl respectively. S3 and S4 culture medium, which have 1/10^th strength of Gamborg's B5 medium, were not suitable for growth of hairy roots. This study also indicated that the salt strength of culture medium (l/4^th or 1/10^th B5) has more effect on growth of root clones than the sucrose concentrations (3% or 1.5%).

For alkaloid extraction, roots were grown in S2 liquid culture medium under similar conditions. Roots were harvested after 35 d and were analyzed for serpentine accumulation. Serpentine accumulation was found to be at the level of 1.8 ± 0.18 mg/g dw which is more than what have been obtained when cultures were grown on solid phase medium (1.12±0.13 mg/g dw). The accumulation of serpentine in this culture condition is much higher than other cases where hairy roots have been generated from other varieties of C roseus (Bhadra and Shanks 1997). This amount of serpentine accumulation is even more than what has been reported recently where hairy roots have been engineered by over-expressing one or more genes of TIA biosynthetic pathway (Hughes et al. 2004; Peebles et al. 2005).

As hairy roots are amenable for scale up in bioreactors, the root clone accumulating high serpentine content can be of commercial use. For alkaloid extraction, roots were grown in liquid culture conditions. Culture conditions were optimized to ¹A strength of B5 culture medium supplemented with 1.5% sucrose for the growth of hairy root clone. 0.6 to 0.8 gm of fresh weight of hairy root was inoculated in 250 ml Erlenmeyer flask containing 40 ml liquid growth medium and was kept on orbital shaker at 80 rpm under 25 + 2°C and 16/8 h light/dark photoperiod. Roots were harvested after 35 d and were dried at room temperature under shade. The dried tissue was homogenized and total alkaloid was extracted. Quantification of alkaloid was done by HPLC analysis.

Serpentine accumulation was found to be at the level of 1.8 ± 0.18 mg/g dw which is more than what have been obtained in other cases where hairy roots have been generated from other varieties of C. roseus (Parr et al. 1988; Bhadra and Shanks 1997). This amount of serpentine accumulation is more than what has been reported earlier where hairy roots have been engineered by overexpressing one or more genes of TIA biosynthetic pathway (Hughes et al. 2004; Peebles et al 2005).

Example 2

Isolation of Serpentine from genetically modified Catharanthus roseus hairy root Genetically modified Catharanthus roseus hairy root (85 g) was cut finely and extracted with methanol. The extracted solution, after evaporation in vacuo, gave a residue (1.67 g) and was evaluated for bioactivity by determining ³H-2DOG uptake in L6 skeletal muscle cells. This fraction was subjected to Diaion HP-20 column chromatography with water and methanol as eluent. The methanol eluent was evaporated to dryness and was further subjected to Sephadex LH-20 column chromatography using methanol-water (1 :1) and methanol as eluent. Evaporation of methanol fraction under reduced pressure yielded a solid (75 mg) that showed biological activity. Preparative HPLC (μ- Bondapak, C- 18 reverse phase column, UV detector at 254nm) of this solid was done using mobile phase consisted of a mixture of 5mM Na₂HPO₄ (pH adjusted to H₃PO₄) (solvent A) and acetonitrile (solvent B); flowrate 2.0 ml /min. The eluent profile (volume of solvent A / volume of solvent B) was (1) 0-20 min: linear gradient from 80:20 (v/v) to 20:80 (v/v); (2) 20-25 min: isocratic elution with 20:80 (v/v) (column rinsing); (3) 25-30 min: isocratic elution with 80:20 (v/v) (column equilibration). A single compound was isolated which showed biological activity. The compound (28 mg) was identified as Serpentine by comparing its physical data as well as its infrared (IR), nuclear magnetic resonance ¹H NMR, ¹³C NMR and mass spectral data with those of as authentic sample.

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Example 3

In vivo treatments

All in-vivo experimental procedures were approved by the Animal Ethics Committee of IICB. Animal house of this institute routinely carries out in-house Sprague-Dawley rat breeding and their maintenance. Male Sprague-Dawley rats weighing approximately 20Og were conditioned at 23+1⁰C with a 12/12 h day/night cycle and fed a standard diet (prepared regularly in this institute for more than 10,000 rats, mice, guinea pigs and hamsters) ad libitum. An insulin resistant obese rat model was produced by a high-fat diet for 4 months by following the method described by Frederic Tremblay et al, Diabetes, 2001. Briefly, as percent of total energy, the high-fat diet consisted of 32.5% lard, 32.5% corn oil, 20% sucrose, and 15% protein, whereas the Rodent Chow diet contained 57.3% carbohydrate, 18.1% protein, and 4.5% fat. The energy contents of the diets were 15 kJ/g for the Rodent Chow diet and 26 kJ/g for the high- fat diet. Each treated and control group contained six male rats with similar range of body weight i.e. between 210-230 g, none of them died during the course of treatment. Feeding of high fat diet for 100 days has significantly increased the body weight in high fat fed group (215+10 g on 0-day to 410 +20 g on 100^th day) as compared to normal diet group (210+25 g on 0-day to 280 + 17 g on 100^th day). Blood was collected for the estimation of the serum glucose level after 12 weeks on a high-fat plus standard chow or only standard chow diet. Glucose levels were determined by enzymatic GOD-POD method, Autospan, Span diagnostics, Surat, India (86+7 mg/dl on 0-day to 175 ± 15 mg/dl on 100^th day) as compared to normal diet group (89+5 mg/dl on 0 day to 92+3 on 100^th day). Insulin levels were also measured by RIA (Radio immunoassay kit, Board of Radiation and isotope technology, Government of India., Department of atomic energy, Mumbai, India) which showed higher amounts in fat fed animals as compared with the control. In-vivo observation was also made in genetically modified type 2 diabetic mice.

Example 4

Cell culture and treatments L6 skeletal muscle cell line was procured from the National Centre for Cell Science, Pune, India and was cultured at 37⁰C in 95%O₂/5% CO₂ in Dulbecco's Modified Eagle's Medium (DMEM) containing 25 mM glucose and 10% fetal calf serum. Confluent cells were treated with 0.75mM palmitate for different time periods as required. Example 5 Cell lysates

Isolated control and treated adult skeletal muscle cell pellets were resuspended in lysis buffer (1% NP-40, 20 mM HEPES (pH 7.4), 2 mM EDTA, 100 mM NaF, 10 mM sodium pyrophosphate, 1 mM sodium orthovanadate, 1 μg/ml leupeptin, 1 μg/ml aprotinin, 1 μg/ml pepstatin and 1 mM PMSF) and sonicated on ice for 10 min. Lysates were centrifuged for 10 min at 10,000g and protein concentrations were determined by the method of Lowry et al.

[³H] Deoxy-glucose uptake

³H Deoxy-glucose uptake in isolated adult rat skeletal muscle cell culture was done according to an earlier described procedure from our laboratory (Roy et al, 2003). Isolated cells were resuspended in Kreb's Ringer Phosphate (KRP) buffer (12.5 mM HEPES, pH 7.4, 120 mM NaCl, 6 mM KCl, 1.2 mM MgSO₄, 1 mM CaCl₂, 0.4 mM NaH₂PO₄, 0.6 mM Na₂HPO₄) supplemented with 0.2 % bovine serum albumin. Cells were incubated with 0.75 mM palmitate for different time periods in the presence or absence of serpentine (20 μg/ml) followed by 30 min incubation with porcine insulin (10OnM) either alone or with palmitate. ³H-deoxyglucose (0.4 nmoles) was added to each incubation mixture 5 min prior to the termination of incubation. Myocytes were washed thrice with ice-cold KRP buffer in the presence of 0.3 mM phloretin to correct the glucose uptake data from simple diffusion and non-specific trapping of radioactivity. Cells were solubilized with 1% NP-40 and [³H]-deoxyglucose was measured in a Liquid Scintillation counter (Packard, Tricarb 2100 TR).

Example 6 Immunoprecipitation

200 μg of control and treated cell lysates were incubated overnight at 4⁰C with 2μg insulin receptor (IR) β antibody. 50 μl of Protein A-agarose was added to_. each tube and incubated at 4⁰C for 2 h. After centrifugation at 10,000 g for 2 min at 4⁰C, 500 μl of 0.1 % CHAPS in PBS was added to the pellets, re-suspended and ^' centrifuged at 10,000 g at 4⁰C for 2 min. The pellets were washed thoroughly, boiled in SDS-PAGE sample buffer and subjected to SDS-PAGE followed by Western Blot using anti-phosphotyrosine antibody (anti-mouse; 1 : 1000). Example 7

Electrophoresis and Immunoblotting

Cell pellets from control and treated incubations were suspended in lysis buffer (1% NP-40, 20 mM HEPES (pH 7.4), 2 mM EDTA, 100 mM NaF, 10 mM sodium pyrophosphate, 1 mM sodium orthovanadate, 1 μg/ml leupeptin, 1 μg/ml aprotinin, 1 μg/ml pepstatin and 1 mM PMSF) and sonicated on ice at 150 KHz for 5 min. Lysates were centrifuged for 10 min at 10,00Og, supernatant was collected and protein content was estimated according to Lowry et al. Control and treated cell lysates (60 μg) were resolved on 10% SDS-PAGE and transferred to PVDF membranes (Millipore, Bedford, MA 01730) through transfer buffer (25 mM Tris, 193 mM glycine, 20 % methanol, pH 8.5) for 1.5 h. Electrophoresis was carried out at 90V constant voltage. Membranes were incubated with 5% Blocking buffer (2OmM Tris base, 137mM NaCl, ImM HCl, 0.1% Tween 20 and 5% non-fat milk) for Ih followed by incubation with primary antibodies such as anti insulin receptor β (IRβ, anti-rabbit), anti IRS 1 (anti-goat), anti p-IRS 1 (anti-goat), anti-PDKl (anti- rabbit), anti-p-PDKl (anti-rabbit), anti-PI3K (anti-rabbit), anti-p-PI3K (anti- rabbit), anti-IκBα (anti-rabbit), anti-IκBα (anti-rabbit), anti-IKK (anti-rabbit), anti- p-IKK (anti -rabbit) antibodies at 1:1000 dilution, overnight at 4⁰C. Bound primary antibodies were visualized using corresponding secondary antibodies at 1 : 1000 dilution, which were tagged either with alkaline phosphatase or horse-radish peroxidase and were developed with corresponding substrates nitro blue tetrazolium / 5-bromo 4-chloro 3-indolyl phosphate (NBT/BCIP). Results obtained with western blots were subjected to densitometric analysis using Imagemaster ID Ellite v3.01 software (Amersham Biosciences, Buckinghamshire, England).

Example 8 RNA Isolation Total RNA was isolated using Tripure™ Isolation Reagent following manufacturer's protocol. To 100 mg of tissue samples 1 ml Tripure was added at room temperature. The tissues were then homogenised using glass-teflon homogeniser. After that the homogenised samples were incubated for 5 minutes at room temperature to ensure the dissociation of nucleoprotein complexes. Thereafter 0.2ml of chloroform was added, shaken vigorously and incubated for 15 minutes at room temperature. After incubation, centrifugation was done at 12000 X g for 15 minutes at 4⁰C (Eppendorf Cold Centrifuge) to separate the solution into 3 phases. The upper aqueous colourless layer was used for isolation of RNA, which was precipitated by addition of 0.5ml isopropanol to the new tube, mixed by inversion and then followed by incubation for 5-10 minutes at room temperature. This was centrifuged at 12000 X g for 10 minutes at 4⁰C. The supernatant was discarded. The pellet was then washed with 75% ethanol and centrifuged at 7500 X g for 5 minutes at 4⁰C. The supernatant was discarded and excess ethanol was removed from RNA pellet by air-drying. Finally the RNA pellet was resuspended in DEPC-treated RNAse-free water by incubating the solution for 10 minutes at 55⁰C. Example 9

Reverse transcription PCR

Total RNA from skeletal muscle cells were isolated in a similar manner as described under Northern blot followed by reverse transcription using RT kit (First strand cDNA synthesis kit, Fermentas Life Sciences, RevertAid™, Hanover, MD, USA). Reverse transcription PCR was carried out according to the manufacturer's instructions to observe NF-κB, insulin receptor gene expression.

Example 10

Real time quantitative PCR

Alteration of NFKB and IR gene expression was observed in semi quantitative RT PCR, and was confirmed by real time quantitative PCR (qPCR) by using iCycler

(BioRad) real time PCR machine and Dynamo SYBR Green real time qPCR kit

(Finzymes, Finland). For real time PCR, 3μg of total RNA from each sample was first reverse transcribed and PCR was performed with gene specific primers in a total volume of 20μl. The real time PCR conditions were as follows: initial activation step (95⁰C-15 min), and cycling step [denaturation 95°C-30sec, annealing at 55°C-30sec, and extension for 72°C-30sec X 40 cycles] followed by melt curve analysis (55⁰C- 60⁰C, 15sec, 40X). A house keeping gene β actin was amplified simultaneously in separate tubes, which acted as an internal control. The CT value was corrected by CT reading of corresponding β actin controls. Data from six determinations (mean± SEM) are expressed as relative expression level. The primers used in real time PCR were the following:

IR sense, 5' -GGATGGTC AGTGTGTGG AGA-3 ' SEQ ID NO. : 1 antisense, 5'-TCGTGAGGTTGTGCTTGTTC-S' SEQ ID NO. :2 β actin sense, 5'TGACGGGGTCACCCACACTGTGCCCATCTAS' SEQ ID NO. :3 antisense, 5'CTAGAAGCATTTGCGGTGGACGATGGAGGGS' SEQ ID NO. :4 The cycle number at which IR transcripts were detectable (CT) was normalized to that of β actin, referred to as ΔCT.

Example 11

CT value calculation

In qPCR, relative quantification was done by comparative Cj value calculation. In this method arithmetic formulas are used to calculate relative expression levels, compared with a calibrator, which can for instance be a control (non-treated) sample.

The amount of target normalized to an endogenous housekeeping gene and relative to the calibrator, is then given by 2^{"ΔA cτ}, where ΔΔ Cf=Δ C_τ (sample) - Δ C_τ

(calibrator), and Δ Cj is the C_T of target gene subtracted from the C_T of the housekeeping gene.

Example 12

Isolation and purification of serpentine and its effects on loss insulin activity due to fatty acid

The GM root extract of Catharanthus roseus was purified by solvent fractionation followed by Diaion-HP20 and Sephadex LH-20 chromatography and finally HPLC reverse phase chromatography. The structure of the purified compound i.e. serpentine was determined by 2D NMR and Mass Spectrometry (Fig l.a). To examine insulin resistance due to FFA L6 skeletal muscle cell culture was performed. Incubation of skeletal muscle cells with insulin stimulated ³H -2-deoxyglucose (2-DOG) uptake to more than 2 fold in the skeletal muscle cells, whereas addition of FFA i.e. palmitate showed impairment of 2-DOG uptake. Palmitate affected more than 2-fold reduction of insulin stimulated 2 DOG uptake. Addition of serpentine in this system blocked the inhibitory effect of palmitate on insulin stimulation significantly (p&lt;0.001). Figure 1 shows the results of this experiment. Example 13

Inhibition of Glut 4 translocation by fatty acid and effect of serpentine thereon

Glut 4 is a glucose transporter protein in the cell. It carries glucose from outside into the cell. Insulin stimulated GFP-Glut 4 translocation to the cell membrane from cytosol was inhibited by palmitate and administration of serpentine withdrew this inhibition. This permitted GFP-Glut4 translocation to the membrane (Fig. 2).

Example 14

Impairment of Insulin signaling defect is ameliorated by serpentine

A 6 h incubation of L6 skeletal muscle cells with palmitate prior to insulin challenge inhibited insulin stimulation of Insulin receptor β (IRβ), IRSl and IRSl associated PD Kinase phosphorylation (Figs. 3.a,b,c). By using phospho-specific antibodies directed against IRS 1 and PI3 Kinase, we have observed that the increase of IRSl and IRSl associated PB Kinase phosphorylation by insulin was totally inhibited by palmitate (Fig.3 a, b). Decrease of IRβ, IRS 1 and IRS 1 associated PD Kinase phosphorylation by FFA has been reported earlier (Griffin ME et al., 1999; Yu C et al., 2002), but what is new in our observation was the removal of palmitate-induced inhibition of insulin activity by serpentine. We then investigated other insulin signal transduction molecules, such as PDKl and Akt/PKB. PDKl is the upstream kinase that directly phosphorylates downstream substrate, Akt/PKB. Insulin stimulated phosphorylation of PDKl and Akt/PKB was inhibited in palmitate incubated L6 skeletal muscle cells. Palmitate did not alter IRSl, PD kinase, PDKl and Akt protein profile of L6 skeletal muscle cells (Fig. 4).

Example 15

Downregulation of insulin receptor gene and protein expression due to fatty acid is significantly blocked by serpentine

Palmitate incubation of skeletal muscle cells for 6h demonstrated decrease in the IR gene transcription as detected by RT-PCR. Incubation of skeletal muscle cells with insulin did not alter Insulin Receptor (IR) gene expression. Addition of serpentine in this incubation blocked the palmitate-induced inhibition of IR gene transcription (Fig.

5a). Real-time PCR data showed a highly significant (p &lt; 0.001) quantitative decrease of IR mRNA due to palmitate (Fig. 5b) which was prevented by serpentine. Consequent reduction of IRβ protein due to the inhibition of IR mRNA is depicted in

Fig. 5c. Serpentine blocks the effect of palmitate induced inhibition of IR gene transcription in L6 skeletal muscle cells.

Example 16

Effect of serpentine on genetically modified diabetic mice The above in-vitro experiments with skeletal muscle cell line clearly indicate that free fatty acid i.e. palmitate, induced decrease of insulin activity, which is the major .symptom in type2 diabetes, is efficiently intervened by serpentine. Hence, serpentine significantly improved the loss of insulin activity due to FFA. To examine this in diabetic mice, leptin receptor knock out genetically modified mice (GM) was selected. These GM diabetic obese mice have consistent high sugar level (between 400-550 mg/dl). Various concentrations in the range of 10 to 50 /100 gm of serpentine were fed along with the normal diet for 15 days. Of these 20 μ/100 gm g/ 100 gm body weight of serpentine was found to be better to reduce the glucose level. The result is shown in Fig 6. The average glucose level in the plasma of these GM diabetic mice in the experimental group was 450 mg/dl and it was reduced significantly (p&lt;0.01) due to the administration of serpentine. Example 16

Effect of hairy root extract on genetically modified diabetic mice

These GM diabetic obese mice have consistent high sugar level (between 400-550 mg/dl). Concentrated hairy root extract comprising approximately 20 μg/ 100 gm body weight of serpentine was fed along with the normal diet for 15 days. The average glucose level in the plasma of these GM diabetic mice in the experimental group was 450 mg/dl and it was reduced significantly (p&lt;0.01) due to the administration of hairy root extract.

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