WO2010004584A2 - Procédé de production d'un composé antidiabétique à partir d'une culture de racine de catharanthus roseus - Google Patents

Procédé de production d'un composé antidiabétique à partir d'une culture de racine de catharanthus roseus Download PDF

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WO2010004584A2
WO2010004584A2 PCT/IN2009/000389 IN2009000389W WO2010004584A2 WO 2010004584 A2 WO2010004584 A2 WO 2010004584A2 IN 2009000389 W IN2009000389 W IN 2009000389W WO 2010004584 A2 WO2010004584 A2 WO 2010004584A2
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serpentine
hairy
hairy root
diabetes
biomass
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PCT/IN2009/000389
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WO2010004584A3 (fr
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Asis Datta
Samir Bhattacharya
C. Bikas Pal
Jayanti Sen
Suman Dasgupta
Anindita Biswas
Jyoti Batra
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Asis Datta
Samir Bhattacharya
Pal C Bikas
Jayanti Sen
Suman Dasgupta
Anindita Biswas
Jyoti Batra
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Publication of WO2010004584A2 publication Critical patent/WO2010004584A2/fr
Publication of WO2010004584A3 publication Critical patent/WO2010004584A3/fr

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    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12P—FERMENTATION OR ENZYME-USING PROCESSES TO SYNTHESISE A DESIRED CHEMICAL COMPOUND OR COMPOSITION OR TO SEPARATE OPTICAL ISOMERS FROM A RACEMIC MIXTURE
    • C12P17/00—Preparation of heterocyclic carbon compounds with only O, N, S, Se or Te as ring hetero atoms
    • C12P17/18—Preparation of heterocyclic carbon compounds with only O, N, S, Se or Te as ring hetero atoms containing at least two hetero rings condensed among themselves or condensed with a common carbocyclic ring system, e.g. rifamycin
    • C12P17/188—Heterocyclic compound containing in the condensed system at least one hetero ring having nitrogen atoms and oxygen atoms as the only ring heteroatoms
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K31/00—Medicinal preparations containing organic active ingredients
    • A61K31/33—Heterocyclic compounds
    • A61K31/395—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins
    • A61K31/435—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins having six-membered rings with one nitrogen as the only ring hetero atom
    • A61K31/47—Quinolines; Isoquinolines
    • A61K31/475—Quinolines; Isoquinolines having an indole ring, e.g. yohimbine, reserpine, strychnine, vinblastine
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K36/00—Medicinal preparations of undetermined constitution containing material from algae, lichens, fungi or plants, or derivatives thereof, e.g. traditional herbal medicines
    • A61K36/18—Magnoliophyta (angiosperms)
    • A61K36/185—Magnoliopsida (dicotyledons)
    • A61K36/24—Apocynaceae (Dogbane family), e.g. plumeria or periwinkle
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P3/00—Drugs for disorders of the metabolism
    • A61P3/08—Drugs for disorders of the metabolism for glucose homeostasis
    • A61P3/10—Drugs for disorders of the metabolism for glucose homeostasis for hyperglycaemia, e.g. antidiabetics

Definitions

  • 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.
  • Plants being static organism, need to cope with the changing environment for its survival, growth and development.
  • 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.
  • TIAs Terpenoid indole alkaloids
  • 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.
  • Typel diabetes or insulin dependent diabetes mellitus is basically due to autoimmune mediated destruction of pancreatic ⁇ islets resulting insulin deficiency.
  • IDDM insulin dependent diabetes mellitus
  • NIDDM non insulin dependent diabetes mellitus
  • FFA free fatty acids
  • HMGAl a High mobility group protein
  • SpI and c/EBP ⁇ two ubiquitously expressed transcription factors
  • SpI and c/EBP ⁇ two ubiquitously expressed transcription factors
  • HMGAl induces transcriptional activation of IR gene by recruiting SpI and c/EBP ⁇ 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/EBP ⁇ 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.
  • NF- ⁇ B NF- ⁇ B kinase
  • IKK inhibitor of KB kinase
  • phosphorylated IKB is degraded releasing NF- ⁇ B for its translocation to the nucleus which regulates number of gene expression.
  • a short term lipid infusion in humans induces skeletal muscle insulin resistance along with a reduction of IKB levels, indicating the activation of NF- ⁇ B during insulin resistance.
  • FFA induced activation of NF- ⁇ B in L6 cell causes insulin resistance, while inhibition of NF- ⁇ B 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.
  • IL-6 interleukin 6
  • TZD thiazolidinediones
  • 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.
  • 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.
  • compositions for treatment of diabetes and insulin resistance comprising a therapeutically effective amount of serpentine and a pharmaceutically or nutritionally acceptable carrier.
  • compositions for treatment of diabetes and insulin resistance 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
  • 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
  • compositions for treatment of diabetes and insulin resistance comprising a Catharanthus roseus hairy root extract, wherein the hairy root extract is prepared by
  • the hairy root extract comprises serpentine in the range of 1.12 to 1.80 mg/g dry weight of the hairy roots.
  • 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
  • IP Insulin stimulated Insulin receptor
  • p-Ty tyrosine kinase
  • p-IRSl phosphorylation of insulin receptor substrate
  • p-PI3K Phosphatidyl Inositol-3 kinase
  • 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.
  • 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.
  • 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.
  • 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.
  • the pharmaceutical preparation disclosed in the present invention can be administered orally in the form of powder, granules or capsule.
  • the dried product can be administered orally in the form of powder, granules or capsule.
  • the fraction is administered at a dose level of 20 ⁇ g/kg body weight for a period 30 days.
  • it relates to a. method for treating animals and human beings.
  • plant material is used for extraction with appropriate solvent such as methanol, halogenated solvent in a percolator or the equipment known in the art.
  • 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 1 H NMR, 13 C NMR and mass spectral data with those of as authentic sample.
  • IR infrared
  • 13 C NMR nuclear magnetic resonance 1 H NMR
  • mass spectral data 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.
  • GM diabetic obese mice have consistent high sugar level (between 400-550 mg/dl). 20 ⁇ g/ 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.
  • One embodiment of the present invention provides the process of production of serpentine in hairy root culture of Catharanthus roseus, the process comprises
  • 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
  • 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.
  • 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 ⁇ g /100 gm body weight to 50 ⁇ g /100 gm body weight of serpentine preferably 20 ⁇ g/100 gm of serpentine.
  • Yet another embodiment of the present invention provides a dosage of the composition comprising serpentine, wherein the dosage comprises 10 ⁇ g /100 gm body weight to 50 ⁇ g /100 gm body weight of serpentine preferably 20 ⁇ g/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.
  • 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 ⁇ g /100 gm body weight to 50 ⁇ g /100 gm body weight of serpentine preferably 20 ⁇ g/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
  • Another embodiment of the present invention provides a process of making Catharanthus roseus hairy root extract, the process comprises
  • 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.
  • the hairy root extract comprises serpentine in the range of 1.12 to 1.80 mg/g dry weight of the hairy roots.
  • composition as disclosed in the present invention elevated tissue fatty acid content when administered to a subject in need thereof.
  • composition as disclosed in the present invention reduces abnormally elevated blood glucose levels when administered to a subject in need thereof.
  • 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 cm 2 ) 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 28 0 C with continuous shaking at 200 rpm until OD 600 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.
  • 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 ' ⁇ B5 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.
  • tissue was dried under shade and dried tissue was homogenized and total alkaloid was extracted. Quantification of alkaloid was done by HPLC analysis.
  • Hairy roots are known as solid phase biocatalyst and not dispersed in liquid like suspended cells.
  • growth of root clones should be optimized in liquid culture medium for biomass accumulation.
  • NCHR-5 which accumulates high amount of serpentine was grown in liquid culture conditions.
  • NCHR-5 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%).
  • the root clone accumulating high serpentine content can be of commercial use.
  • roots were grown in liquid culture conditions. Culture conditions were optimized to 1 A 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).
  • 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).
  • the compound (28 mg) was identified as Serpentine by comparing its physical data as well as its infrared (IR), nuclear magnetic resonance 1 H NMR, 13 C NMR and mass spectral data with those of as authentic sample.
  • the high-fat diet consisted of 32.5% lard, 32.5% corn oil, 20% sucrose, and 15% protein
  • 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.
  • L6 skeletal muscle cell line was procured from the National Centre for Cell Science, Pune, India and was cultured at 37 0 C in 95%O 2 /5% CO 2 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.
  • DMEM Dulbecco's Modified Eagle's Medium
  • 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 ⁇ g/ml leupeptin, 1 ⁇ g/ml aprotinin, 1 ⁇ g/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.
  • 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 ⁇ g/ml leupeptin, 1 ⁇ g/ml aprotinin, 1 ⁇ g/ml pepstatin and 1 mM PMSF
  • 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 ⁇ g/ml leupeptin, 1 ⁇ g/ml aprotinin, 1 ⁇ g/ml pepstatin and 1 mM PMSF
  • 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 ⁇ g 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 ⁇ (IR ⁇ , 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-I ⁇ B ⁇ (anti-rabbit), anti-I ⁇ B ⁇ (anti-rabbit), anti-IKK (anti-rabbit), anti- p-IKK (anti -rabbit) antibodies at 1:1000 dilution, overnight at 4 0 C.
  • Blocking buffer 2OmM Tris base, 137mM NaCl, ImM HCl, 0.1% Tween 20 and 5% non-fat milk
  • primary antibodies such as
  • 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).
  • TripureTM 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
  • RNA 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 4 0 C. The supernatant was discarded. The pellet was then washed with 75% ethanol and centrifuged at 7500 X g for 5 minutes at 4 0 C. 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 55 0 C.
  • 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, RevertAidTM, Hanover, MD, USA). Reverse transcription PCR was carried out according to the manufacturer's instructions to observe NF- ⁇ B, insulin receptor gene expression.
  • RT kit First strand cDNA synthesis kit, Fermentas Life Sciences, RevertAidTM, Hanover, MD, USA.
  • Reverse transcription PCR was carried out according to the manufacturer's instructions to observe NF- ⁇ B, insulin receptor gene expression.
  • RNA from each sample was first reverse transcribed and PCR was performed with gene specific primers in a total volume of 20 ⁇ l.
  • the real time PCR conditions were as follows: initial activation step (95 0 C-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 (55 0 C- 60 0 C, 15sec, 40X).
  • a house keeping gene ⁇ actin was amplified simultaneously in separate tubes, which acted as an internal control.
  • the CT value was corrected by CT reading of corresponding ⁇ 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
  • ⁇ actin sense 5'TGACGGGGTCACCCACACTGTGCCCATCTAS' SEQ ID NO. :3 antisense, 5'CTAGAAGCATTTGCGGTGGACGATGGAGGGS' SEQ ID NO. :4
  • CT cycle number at which IR transcripts were detectable
  • ⁇ Cj is the C T of target gene subtracted from the C T of the housekeeping gene.
  • 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 3 H -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 ⁇ 0.001). Figure 1 shows the results of this experiment.
  • 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).
  • 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).
  • 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.
  • Fig. 5c Serpentine blocks the effect of palmitate induced inhibition of IR gene transcription in L6 skeletal muscle cells.
  • serpentine significantly improved the loss of insulin activity due to FFA.
  • leptin receptor knock out genetically modified mice was selected. These GM diabetic obese mice have consistent high sugar level (between 400-550 mg/dl).
  • concentrations in the range of 10 to 50 /100 gm of serpentine were fed along with the normal diet for 15 days.
  • GM diabetic obese mice have consistent high sugar level (between 400-550 mg/dl).
  • Concentrated hairy root extract comprising approximately 20 ⁇ g/ 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 hairy root extract.

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Abstract

L'invention concerne le traitement du diabète au moyen de serpentine. L'invention concerne plus particulièrement un procédé de production de serpentine dans une culture de racines chevelues de Catharanthus roseus, et l'effet d'un extrait de racine chevelue sur la réduction de glucose sanguin chez un sujet. L'invention concerne enfin l'identification et l'isolation à partir de l'extrait d'un composé biologiquement actif.
PCT/IN2009/000389 2008-07-09 2009-07-09 Procédé de production d'un composé antidiabétique à partir d'une culture de racine de catharanthus roseus WO2010004584A2 (fr)

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CN102613088A (zh) * 2012-04-23 2012-08-01 上海安体康生植物化学有限公司 培育耐盐耐低温长春花株系快速繁殖的方法
WO2015095625A1 (fr) * 2013-12-19 2015-06-25 Rhizoflora, Inc. Composition d'activateur de plantes
CN111500625A (zh) * 2020-05-18 2020-08-07 扬州大学 一种利用发根农杆菌诱导产生玫瑰毛状根的方法

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BHADRA R ET AL: "Production of indole alkaloids by selected hairy root lines of Catharanthus roseus." BIOTECHNOLOGY AND BIOENGINEERING 5 MAR 1993, vol. 41, no. 5, 5 March 1993 (1993-03-05), pages 581-592, XP002565273 ISSN: 0006-3592 *
BHADRA R ET AL: "Transient studies of nutrient uptake, growth, and indole alkaloid accumulation in heterotrophic cultures of hairy roots of Catharanthus roseus." BIOTECHNOLOGY AND BIOENGINEERING 5 AUG 1997, vol. 55, no. 3, 5 August 1997 (1997-08-05), pages 527-534, XP002565274 ISSN: 0006-3592 cited in the application *
PARKER JANICE C: "Troglitazone: the discovery and development of a novel therapy for the treatment of Type 2 diabetes mellitus" ADVANCED DRUG DELIVERY REVIEWS 5 NOV 2002,, vol. 54, no. 9, 5 November 2002 (2002-11-05), pages 1173-1197, XP002562968 *
PEEBLES CHRISTIE A M ET AL: "Effects of terpenoid precursor feeding on Catharanthus roseus hairy roots over-expressing the alpha or the alpha and beta subunits of anthranilate synthase" BIOTECHNOLOGY AND BIOENGINEERING, vol. 93, no. 3, February 2006 (2006-02), pages 534-540, XP002565276 ISSN: 0006-3592 *
SCRAGG A H ET AL: "Effect of scale-up on serpentine formation by Catharanthus roseus suspension cultures" ENZYME AND MICROBIAL TECHNOLOGY, STONEHAM, MA, US, vol. 9, no. 10, 1 October 1987 (1987-10-01), pages 619-624, XP023679097 ISSN: 0141-0229 [retrieved on 1987-10-01] *
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Cited By (5)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN102613088A (zh) * 2012-04-23 2012-08-01 上海安体康生植物化学有限公司 培育耐盐耐低温长春花株系快速繁殖的方法
WO2015095625A1 (fr) * 2013-12-19 2015-06-25 Rhizoflora, Inc. Composition d'activateur de plantes
US10287609B2 (en) 2013-12-19 2019-05-14 Rhizoflora Inc. Plant activator composition
US10612046B2 (en) 2013-12-19 2020-04-07 Rhizoflora Inc. Plant activator composition
CN111500625A (zh) * 2020-05-18 2020-08-07 扬州大学 一种利用发根农杆菌诱导产生玫瑰毛状根的方法

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