WO2007042902A2 - Extracts from nyctanthes arbortristis for the treatement of leishmaniasis - Google Patents
Extracts from nyctanthes arbortristis for the treatement of leishmaniasis Download PDFInfo
- Publication number
- WO2007042902A2 WO2007042902A2 PCT/IB2006/002805 IB2006002805W WO2007042902A2 WO 2007042902 A2 WO2007042902 A2 WO 2007042902A2 IB 2006002805 W IB2006002805 W IB 2006002805W WO 2007042902 A2 WO2007042902 A2 WO 2007042902A2
- Authority
- WO
- WIPO (PCT)
- Prior art keywords
- body weight
- composition
- bioactive fraction
- piperine
- unit dose
- Prior art date
Links
- 240000005199 Nyctanthes arbor tristis Species 0.000 title claims abstract description 34
- 235000005215 Nyctanthes arbor tristis Nutrition 0.000 title claims abstract description 19
- 239000000284 extract Substances 0.000 title claims abstract description 11
- 208000004554 Leishmaniasis Diseases 0.000 title description 6
- 230000000975 bioactive effect Effects 0.000 claims abstract description 166
- 238000000034 method Methods 0.000 claims abstract description 51
- UHIGZYLCYRQESL-UHFFFAOYSA-N Desrhamnosylacteoside Natural products OC1C(O)C(OC(=O)C=CC=2C=C(O)C(O)=CC=2)C(CO)OC1OCCC1=CC=C(O)C(O)=C1 UHIGZYLCYRQESL-UHFFFAOYSA-N 0.000 claims abstract description 35
- 206010047505 Visceral leishmaniasis Diseases 0.000 claims abstract description 35
- UHIGZYLCYRQESL-VJWFJHQPSA-N calceolarioside A Chemical compound O([C@@H]1O[C@@H]([C@H]([C@H](O)[C@H]1O)OC(=O)\C=C\C=1C=C(O)C(O)=CC=1)CO)CCC1=CC=C(O)C(O)=C1 UHIGZYLCYRQESL-VJWFJHQPSA-N 0.000 claims abstract description 35
- 210000000952 spleen Anatomy 0.000 claims abstract description 34
- 210000004185 liver Anatomy 0.000 claims abstract description 32
- 230000000724 leishmaniacidal effect Effects 0.000 claims abstract description 17
- 238000011282 treatment Methods 0.000 claims abstract description 17
- 238000002955 isolation Methods 0.000 claims abstract description 8
- 231100000252 nontoxic Toxicity 0.000 claims abstract description 5
- 230000003000 nontoxic effect Effects 0.000 claims abstract description 5
- 230000037396 body weight Effects 0.000 claims description 150
- 239000000203 mixture Substances 0.000 claims description 107
- YQDGWZZYGYKDLR-UZVLBLASSA-K sodium stibogluconate Chemical compound O.O.O.O.O.O.O.O.O.[Na+].[Na+].[Na+].O1[C@H]([C@H](O)CO)[C@H](O2)[C@H](C([O-])=O)O[Sb]21([O-])O[Sb]1(O)(O[C@H]2C([O-])=O)O[C@H]([C@H](O)CO)[C@@H]2O1 YQDGWZZYGYKDLR-UZVLBLASSA-K 0.000 claims description 84
- 229960001567 sodium stibogluconate Drugs 0.000 claims description 84
- MXXWOMGUGJBKIW-YPCIICBESA-N piperine Chemical compound C=1C=C2OCOC2=CC=1/C=C/C=C/C(=O)N1CCCCC1 MXXWOMGUGJBKIW-YPCIICBESA-N 0.000 claims description 65
- 229940075559 piperine Drugs 0.000 claims description 65
- WVWHRXVVAYXKDE-UHFFFAOYSA-N piperine Natural products O=C(C=CC=Cc1ccc2OCOc2c1)C3CCCCN3 WVWHRXVVAYXKDE-UHFFFAOYSA-N 0.000 claims description 65
- 235000019100 piperine Nutrition 0.000 claims description 65
- 230000003071 parasitic effect Effects 0.000 claims description 36
- 239000008194 pharmaceutical composition Substances 0.000 claims description 36
- 150000001875 compounds Chemical class 0.000 claims description 24
- 239000000654 additive Substances 0.000 claims description 22
- OKKJLVBELUTLKV-UHFFFAOYSA-N Methanol Chemical compound OC OKKJLVBELUTLKV-UHFFFAOYSA-N 0.000 claims description 18
- 239000003085 diluting agent Substances 0.000 claims description 18
- 239000003937 drug carrier Substances 0.000 claims description 18
- 239000003795 chemical substances by application Substances 0.000 claims description 14
- HEDRZPFGACZZDS-UHFFFAOYSA-N Chloroform Chemical compound ClC(Cl)Cl HEDRZPFGACZZDS-UHFFFAOYSA-N 0.000 claims description 8
- LFQSCWFLJHTTHZ-UHFFFAOYSA-N Ethanol Chemical compound CCO LFQSCWFLJHTTHZ-UHFFFAOYSA-N 0.000 claims description 8
- 150000001720 carbohydrates Chemical class 0.000 claims description 8
- 235000014633 carbohydrates Nutrition 0.000 claims description 8
- UBSCDKPKWHYZNX-UHFFFAOYSA-N Demethoxycapillarisin Natural products C1=CC(O)=CC=C1OC1=CC(=O)C2=C(O)C=C(O)C=C2O1 UBSCDKPKWHYZNX-UHFFFAOYSA-N 0.000 claims description 6
- LRHPLDYGYMQRHN-UHFFFAOYSA-N N-Butanol Chemical compound CCCCO LRHPLDYGYMQRHN-UHFFFAOYSA-N 0.000 claims description 6
- 229930182470 glycoside Natural products 0.000 claims description 6
- 238000000338 in vitro Methods 0.000 claims description 6
- MWDZOUNAPSSOEL-UHFFFAOYSA-N kaempferol Natural products OC1=C(C(=O)c2cc(O)cc(O)c2O1)c3ccc(O)cc3 MWDZOUNAPSSOEL-UHFFFAOYSA-N 0.000 claims description 6
- 235000008777 kaempferol Nutrition 0.000 claims description 6
- -1 kaempferol glycoside Chemical class 0.000 claims description 6
- IYRMWMYZSQPJKC-UHFFFAOYSA-N kaempherol Natural products C1=CC(O)=CC=C1C1=C(O)C(=O)C2=C(O)C=C(O)C=C2O1 IYRMWMYZSQPJKC-UHFFFAOYSA-N 0.000 claims description 6
- 239000000314 lubricant Substances 0.000 claims description 6
- UXOUKMQIEVGVLY-UHFFFAOYSA-N morin Natural products OC1=CC(O)=CC(C2=C(C(=O)C3=C(O)C=C(O)C=C3O2)O)=C1 UXOUKMQIEVGVLY-UHFFFAOYSA-N 0.000 claims description 6
- 239000000843 powder Substances 0.000 claims description 6
- OYPRJOBELJOOCE-UHFFFAOYSA-N Calcium Chemical compound [Ca] OYPRJOBELJOOCE-UHFFFAOYSA-N 0.000 claims description 4
- FYYHWMGAXLPEAU-UHFFFAOYSA-N Magnesium Chemical compound [Mg] FYYHWMGAXLPEAU-UHFFFAOYSA-N 0.000 claims description 4
- 239000011575 calcium Substances 0.000 claims description 4
- 229910052791 calcium Inorganic materials 0.000 claims description 4
- 239000001913 cellulose Substances 0.000 claims description 4
- 229920002678 cellulose Polymers 0.000 claims description 4
- 239000008273 gelatin Substances 0.000 claims description 4
- 229920000159 gelatin Polymers 0.000 claims description 4
- 238000007912 intraperitoneal administration Methods 0.000 claims description 4
- 239000011777 magnesium Substances 0.000 claims description 4
- 229910052749 magnesium Inorganic materials 0.000 claims description 4
- 239000003960 organic solvent Substances 0.000 claims description 4
- 108090000623 proteins and genes Proteins 0.000 claims description 4
- 102000004169 proteins and genes Human genes 0.000 claims description 4
- 239000007787 solid Substances 0.000 claims description 4
- 239000000454 talc Substances 0.000 claims description 4
- 229910052623 talc Inorganic materials 0.000 claims description 4
- VYPSYNLAJGMNEJ-UHFFFAOYSA-N Silicium dioxide Chemical compound O=[Si]=O VYPSYNLAJGMNEJ-UHFFFAOYSA-N 0.000 claims description 3
- 239000000741 silica gel Substances 0.000 claims description 3
- 229910002027 silica gel Inorganic materials 0.000 claims description 3
- 241000124008 Mammalia Species 0.000 claims description 2
- 239000002775 capsule Substances 0.000 claims description 2
- 238000004587 chromatography analysis Methods 0.000 claims description 2
- 239000012141 concentrate Substances 0.000 claims description 2
- 235000008504 concentrate Nutrition 0.000 claims description 2
- 239000008187 granular material Substances 0.000 claims description 2
- 239000001963 growth medium Substances 0.000 claims description 2
- 238000007918 intramuscular administration Methods 0.000 claims description 2
- 238000001990 intravenous administration Methods 0.000 claims description 2
- 210000003734 kidney Anatomy 0.000 claims description 2
- 239000006188 syrup Substances 0.000 claims description 2
- 235000020357 syrup Nutrition 0.000 claims description 2
- 238000004809 thin layer chromatography Methods 0.000 claims description 2
- 230000035899 viability Effects 0.000 claims description 2
- 230000000996 additive effect Effects 0.000 claims 2
- 241000222727 Leishmania donovani Species 0.000 abstract description 22
- 241000699673 Mesocricetus auratus Species 0.000 abstract description 21
- 230000002514 anti-leishmanial effect Effects 0.000 abstract description 11
- 235000012001 Cestrum nocturnum Nutrition 0.000 abstract description 10
- 239000000401 methanolic extract Substances 0.000 abstract description 9
- 238000002648 combination therapy Methods 0.000 abstract description 7
- 238000001727 in vivo Methods 0.000 abstract description 7
- 230000001684 chronic effect Effects 0.000 abstract description 6
- 239000003814 drug Substances 0.000 abstract description 6
- 241000699800 Cricetinae Species 0.000 abstract description 4
- 208000030852 Parasitic disease Diseases 0.000 abstract description 3
- 230000003816 axenic effect Effects 0.000 abstract description 3
- 238000011097 chromatography purification Methods 0.000 abstract description 3
- 238000001514 detection method Methods 0.000 abstract description 3
- 208000009182 Parasitemia Diseases 0.000 abstract description 2
- 238000012512 characterization method Methods 0.000 abstract 1
- 230000002195 synergetic effect Effects 0.000 abstract 1
- IAZDPXIOMUYVGZ-UHFFFAOYSA-N Dimethylsulphoxide Chemical compound CS(C)=O IAZDPXIOMUYVGZ-UHFFFAOYSA-N 0.000 description 33
- 241001465754 Metazoa Species 0.000 description 17
- 208000015181 infectious disease Diseases 0.000 description 17
- 230000000694 effects Effects 0.000 description 13
- 244000045947 parasite Species 0.000 description 10
- 239000000287 crude extract Substances 0.000 description 9
- 238000011553 hamster model Methods 0.000 description 9
- 241000196324 Embryophyta Species 0.000 description 8
- 238000002474 experimental method Methods 0.000 description 7
- 239000002904 solvent Substances 0.000 description 7
- DDRJAANPRJIHGJ-UHFFFAOYSA-N creatinine Chemical compound CN1CC(=O)NC1=N DDRJAANPRJIHGJ-UHFFFAOYSA-N 0.000 description 4
- 201000010099 disease Diseases 0.000 description 4
- 208000037265 diseases, disorders, signs and symptoms Diseases 0.000 description 4
- 238000004128 high performance liquid chromatography Methods 0.000 description 4
- XEKOWRVHYACXOJ-UHFFFAOYSA-N Ethyl acetate Chemical compound CCOC(C)=O XEKOWRVHYACXOJ-UHFFFAOYSA-N 0.000 description 3
- 230000005526 G1 to G0 transition Effects 0.000 description 3
- 210000004369 blood Anatomy 0.000 description 3
- 239000008280 blood Substances 0.000 description 3
- 229940079593 drug Drugs 0.000 description 3
- 230000001575 pathological effect Effects 0.000 description 3
- 210000002966 serum Anatomy 0.000 description 3
- 239000000243 solution Substances 0.000 description 3
- 238000012360 testing method Methods 0.000 description 3
- 231100000419 toxicity Toxicity 0.000 description 3
- 230000001988 toxicity Effects 0.000 description 3
- 108010082126 Alanine transaminase Proteins 0.000 description 2
- 102000002260 Alkaline Phosphatase Human genes 0.000 description 2
- 108020004774 Alkaline Phosphatase Proteins 0.000 description 2
- 108090000790 Enzymes Proteins 0.000 description 2
- 102000004190 Enzymes Human genes 0.000 description 2
- 238000005481 NMR spectroscopy Methods 0.000 description 2
- XSQUKJJJFZCRTK-UHFFFAOYSA-N Urea Chemical compound NC(N)=O XSQUKJJJFZCRTK-UHFFFAOYSA-N 0.000 description 2
- 239000002246 antineoplastic agent Substances 0.000 description 2
- 238000003556 assay Methods 0.000 description 2
- 239000002034 butanolic fraction Substances 0.000 description 2
- 239000004202 carbamide Substances 0.000 description 2
- 229940109239 creatinine Drugs 0.000 description 2
- 229940127089 cytotoxic agent Drugs 0.000 description 2
- 231100000673 dose–response relationship Toxicity 0.000 description 2
- 229940000406 drug candidate Drugs 0.000 description 2
- 238000010828 elution Methods 0.000 description 2
- 239000012091 fetal bovine serum Substances 0.000 description 2
- 239000000706 filtrate Substances 0.000 description 2
- 231100000331 toxic Toxicity 0.000 description 2
- 230000002588 toxic effect Effects 0.000 description 2
- 230000009278 visceral effect Effects 0.000 description 2
- XLYOFNOQVPJJNP-UHFFFAOYSA-N water Chemical compound O XLYOFNOQVPJJNP-UHFFFAOYSA-N 0.000 description 2
- 238000001644 13C nuclear magnetic resonance spectroscopy Methods 0.000 description 1
- PSOYNZJMRVQTDR-UHFFFAOYSA-N Arbortristoside A Natural products C12C(C)C(OC(=O)C=CC=3C=CC(OC)=CC=3)C(O)C2C(C(=O)OC)=COC1OC1OC(CO)C(O)C(O)C1O PSOYNZJMRVQTDR-UHFFFAOYSA-N 0.000 description 1
- 108091003079 Bovine Serum Albumin Proteins 0.000 description 1
- 208000003322 Coinfection Diseases 0.000 description 1
- 206010011224 Cough Diseases 0.000 description 1
- 206010011668 Cutaneous leishmaniasis Diseases 0.000 description 1
- 241000222722 Leishmania <genus> Species 0.000 description 1
- 241000131784 Nyctanthes Species 0.000 description 1
- 101710098398 Probable alanine aminotransferase, mitochondrial Proteins 0.000 description 1
- 206010037660 Pyrexia Diseases 0.000 description 1
- 208000004078 Snake Bites Diseases 0.000 description 1
- 230000009471 action Effects 0.000 description 1
- 238000004458 analytical method Methods 0.000 description 1
- 238000010171 animal model Methods 0.000 description 1
- 230000000507 anthelmentic effect Effects 0.000 description 1
- 230000000840 anti-viral effect Effects 0.000 description 1
- 229940124573 antileishmanial agent Drugs 0.000 description 1
- 239000000045 antileishmanial agent Substances 0.000 description 1
- 238000013459 approach Methods 0.000 description 1
- SESFRYSPDFLNCH-UHFFFAOYSA-N benzyl benzoate Chemical compound C=1C=CC=CC=1C(=O)OCC1=CC=CC=C1 SESFRYSPDFLNCH-UHFFFAOYSA-N 0.000 description 1
- YBHILYKTIRIUTE-UHFFFAOYSA-N berberine Chemical compound C1=C2CC[N+]3=CC4=C(OC)C(OC)=CC=C4C=C3C2=CC2=C1OCO2 YBHILYKTIRIUTE-UHFFFAOYSA-N 0.000 description 1
- 229940093265 berberine Drugs 0.000 description 1
- QISXPYZVZJBNDM-UHFFFAOYSA-N berberine Natural products COc1ccc2C=C3N(Cc2c1OC)C=Cc4cc5OCOc5cc34 QISXPYZVZJBNDM-UHFFFAOYSA-N 0.000 description 1
- 210000001185 bone marrow Anatomy 0.000 description 1
- 238000005119 centrifugation Methods 0.000 description 1
- 238000002512 chemotherapy Methods 0.000 description 1
- 238000013329 compounding Methods 0.000 description 1
- 238000011161 development Methods 0.000 description 1
- 238000009509 drug development Methods 0.000 description 1
- 208000001848 dysentery Diseases 0.000 description 1
- 238000011156 evaluation Methods 0.000 description 1
- 238000000605 extraction Methods 0.000 description 1
- 229940124600 folk medicine Drugs 0.000 description 1
- 238000009472 formulation Methods 0.000 description 1
- 238000005194 fractionation Methods 0.000 description 1
- 230000005802 health problem Effects 0.000 description 1
- 206010019847 hepatosplenomegaly Diseases 0.000 description 1
- 238000000099 in vitro assay Methods 0.000 description 1
- 230000005764 inhibitory process Effects 0.000 description 1
- 239000007924 injection Substances 0.000 description 1
- 238000002347 injection Methods 0.000 description 1
- 230000000968 intestinal effect Effects 0.000 description 1
- 239000007928 intraperitoneal injection Substances 0.000 description 1
- 229930182489 iridoid glycoside Natural products 0.000 description 1
- 150000008145 iridoid glycosides Chemical class 0.000 description 1
- 239000007788 liquid Substances 0.000 description 1
- 238000004949 mass spectrometry Methods 0.000 description 1
- 239000000463 material Substances 0.000 description 1
- 239000002609 medium Substances 0.000 description 1
- 229930014626 natural product Natural products 0.000 description 1
- 230000001766 physiological effect Effects 0.000 description 1
- 238000012746 preparative thin layer chromatography Methods 0.000 description 1
- 230000008569 process Effects 0.000 description 1
- 230000000750 progressive effect Effects 0.000 description 1
- 244000000040 protozoan parasite Species 0.000 description 1
- 238000000746 purification Methods 0.000 description 1
- 239000002994 raw material Substances 0.000 description 1
- 206010040882 skin lesion Diseases 0.000 description 1
- 231100000444 skin lesion Toxicity 0.000 description 1
- 239000011734 sodium Substances 0.000 description 1
- 238000001228 spectrum Methods 0.000 description 1
- 239000008223 sterile water Substances 0.000 description 1
- 238000004659 sterilization and disinfection Methods 0.000 description 1
- 239000011550 stock solution Substances 0.000 description 1
- 238000003239 susceptibility assay Methods 0.000 description 1
- 239000000725 suspension Substances 0.000 description 1
- 230000009885 systemic effect Effects 0.000 description 1
- 239000012085 test solution Substances 0.000 description 1
- 230000001225 therapeutic effect Effects 0.000 description 1
- 238000002560 therapeutic procedure Methods 0.000 description 1
- 231100000041 toxicology testing Toxicity 0.000 description 1
- 238000011269 treatment regimen Methods 0.000 description 1
- 229960005486 vaccine Drugs 0.000 description 1
- 238000005303 weighing Methods 0.000 description 1
Classifications
-
- 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)
- A61K36/63—Oleaceae (Olive family), e.g. jasmine, lilac or ash tree
-
- 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
- A61K31/44—Non condensed pyridines; Hydrogenated derivatives thereof
- A61K31/445—Non condensed piperidines, e.g. piperocaine
- A61K31/4453—Non condensed piperidines, e.g. piperocaine only substituted in position 1, e.g. propipocaine, diperodon
-
- 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
- A61K31/555—Heterocyclic compounds containing heavy metals, e.g. hemin, hematin, melarsoprol
-
- 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
- A61K31/70—Carbohydrates; Sugars; Derivatives thereof
- A61K31/7028—Compounds having saccharide radicals attached to non-saccharide compounds by glycosidic linkages
-
- 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
- A61K31/70—Carbohydrates; Sugars; Derivatives thereof
- A61K31/7042—Compounds having saccharide radicals and heterocyclic rings
- A61K31/7048—Compounds having saccharide radicals and heterocyclic rings having oxygen as a ring hetero atom, e.g. leucoglucosan, hesperidin, erythromycin, nystatin, digitoxin or digoxin
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
- A61P33/00—Antiparasitic agents
- A61P33/02—Antiprotozoals, e.g. for leishmaniasis, trichomoniasis, toxoplasmosis
-
- Y—GENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
- Y02—TECHNOLOGIES OR APPLICATIONS FOR MITIGATION OR ADAPTATION AGAINST CLIMATE CHANGE
- Y02A—TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE
- Y02A50/00—TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE in human health protection, e.g. against extreme weather
- Y02A50/30—Against vector-borne diseases, e.g. mosquito-borne, fly-borne, tick-borne or waterborne diseases whose impact is exacerbated by climate change
Definitions
- the present invention relates to a bioactive fraction obtained from the leaves of the plant night jasmine.
- bioactive fraction in the treatment of visceral leshmaniasis.
- the present invention also relates to the use of the compound calceolarioside A as a leishmanicidal agent.
- compositions useful as a leishmanicidal agent comprising the therapeutically effective amount of said bioactive fraction obtained from the leaves of the plant night jasmine or selected from the combination comprising the bioactive fraction and sodium antimony gluconate or bioactive fraction and piperine or bioactive fraction and sodium antimony gluconate and piperine optionally along with one or more pharmaceutically acceptable carriers, additives, lubricant and diluents.
- the present invention also relates to a method for the isolation of compound calceolarioside A from the bioactive fraction obtained from the leaves of the plant
- Nyctanthes arbortristis is a traditionally used medicinal plant of India commonly used as an anthelmintic, antibilious, diaphoretic etc. and fpr the treatment of various diseases such as fever, rheumatism, intestinal worm disinfections etc. (The Wealth of India, Raw Materials, (1966) Vol. 7, pp 69-70). Tribal people of central India used various parts of this plant for relieving cough, dysentery, snakebite, sores etc. (S.KJain, Dictionary of Indian folk medicine and Ethnobotany, 1991). Most of the recent bioactivity studies on N.
- arbortristis has been carried out from seed extract and iridoid glycosides (arbortristoside A, B, C, D, E) were isolated which showed antiviral (Anita Rathore, Vandita Srivastava, K.C.Srivastava and J.S. Tandon, Phytochemistry (1990) Vol.29 pp.1917-1920) or antileishmanial activity (J.S.Tandon, Vandita Srivastava and P.Y.Guru, Journal of Natural Products, (1991) Vol.54 pp.l 102-1104).
- calceolarioside A a new class of compound to be reported from this plant, was isolated and purified from the methanolic extract of night jasmine leaf and showed significant antileishmanial activity both in vitro and in vivo test systems. This is the first isolation report of calceolarioside A from this plant as well as the first antileishmanial activity report of calceolarioside A.
- the main object of the present invention is to provide a bioactive fraction obtained from the leaves of the plant night jasmine.
- Another object of the present invention is to provide the use of said bioactive fraction in the treatment of visceral leishmaniasis. Further object of the present invention is to provide the use of the compound calceolarioside A as a leishmanicidal agent.
- the present invention deals with a bioactive fraction obtained from the leaves of the plant night jasmine comprising a) calceolarioside A b) unidentified compounds c) kaempferol glycoside. It also provides the use of the said fraction and the compound calceolarioside A in the treatment of visceral leishmaniasis. Further, it also relates to a pharmaceutical composition and method of treating visceral leishmaniasis using said composition. The invention also relates to a method of isolation of said compound from the bioactive fraction obtained from the leaves of the plant Nyctanthes arbortristis.
- Figure 1 represents the effect of crude methanolic extract of Nyctanthes arbortristis leaf and PE on the growth of Leishmania donovani promastigotes in vitro in time and dose dependent manner. Viable cells, i.e., promastigotes (xlO 6 )/nil of culture. Data are shown for one representative experiment out of three with similar results. Solvent was DMSO+PBS.
- Figure 2 represents the effect of Nyctanthes arbortristis crude methanolic leaf extract against established infection of visceral leishmaniasis in golden hamster model.
- Two weeks infected golden hamsters were treated with crude extract at the dose of lOmg/kg, 20mg/kg, 40 mg/kg and 60mg/kg body weight for 21 days. Controls were infected untreated animals. Crude extract was administered through i.p route. Treated animals received total six shots of crude extract during three weeks of treatment schedule. Animals were sacrificed after six weeks of infection. Levels of parasite burden in spleen and liver are expressed in Leishman-Donovan units (LDU).
- LDU Leishman-Donovan units
- Figure 3 represents the effect of methanolic extract of Nyctanthes arhortritis leaf and SAG in established infection of Leishmania donovani.
- Four weeks infected golden hamsters were treated intra-peritoneally with crude methanolic extract at the doses of 10mg/kg body weight, 20 mg/kg body weight and SAG 10mg/kg body weight for 7 days respectively.
- Controls were infected untreated animals.
- Treated animals received one shot in one week to complete the treatment.
- Level of parasite burden in spleen is expressed in Leishman-Donovan units (LDU).
- DMSO: PBS DMSO+PBS as solvent of crude extract
- Figure 4 represents the effect of bioactive fraction and combined therapy of bioactive fraction + SAG in established infection of visceral leishmaniasis.
- Viable cells i.e., promastigotes (xlO 6 )/ml of culture. Data are shown for one representative experiment out of three with similar results.
- Figure 7 represents the effect of bioactive fraction and combined therapy of bioactive fraction, SAG and piperine against established infection of visceral leishmaniasis in golden hamster model. Effect of bioactive fraction, combined therapy of bioactive fraction + piperine, bioactive fraction + SAG and bioactive fraction + SAG + piperine against established infection of visceral leishmaniasis in golden hamster model.
- Fig 8 HPLC chromatogram of bioactive fraction obtained from methanolic extract of night jasmine leaf.
- Fig 9 HPLC chromatogram of calceolarioside A obtained from methanolic extract of night jasmine leaf. Detailed description of the invention:
- the present invention provides a bioactive fraction obtained from the leaves of the plant night jasmine, wherein the said fraction comprising a) calceolarioside A 39.62% b) kaempferol glycoside 34.8%, c) two unidentified compound 5.2% & 5.8% and d) unidentified unresolved fraction 14.6%, wherein said weight percentages are percentages in the total weight of the fraction.
- the said fraction is useful as a leishmanicidal agent.
- the said fraction is non toxic to liver and kidney at the dose of 200mg/Kg body weight / day at least for three weeks.
- the present invention also provides the use of the bioactive fraction in the treatment of visceral leshmaniasis.
- the compound calceolarioside A as a leishmanicidal agent.
- the said compound reduces the viability of
- L. donovani promastigotes in vitro uptolOO % at the dose of 80 ⁇ g/ml of culture media for 24 hrs.
- the present invention also provides a pharmaceutical composition useful as a leishmanicidal agent, wherein the said composition comprising the therapeutically effective amount of said bioactive fraction obtained from the leaves of the plant night jasmine or selected from the combination comprising the bioactive fraction and sodium antimony gluconate or bioactive fraction and piperine or bioactive fraction and sodium antimony gluconate and piperine optionally along with one or more pharmaceutically acceptable carriers, additives, lubricant and diluents.
- the additives used are selected from the group comprising of proteins, carbohydrates, sugar, talc, magnesium state, cellulose, calcium, carbohydrate, starch-gelatin paste.
- the said composition further comprising the therapeutically effective amount of bioactive fraction obtained from the leaves of the plant night jasmine optionally along with one or more pharmaceutically acceptable carriers, additives and diluents.
- the dosage of the said composition is administered at a unit dose of at least 100 to 200 mg/Kg body weight.
- the said composition is preferably administered at a unit dose of 100 mg/Kg body weight at least for three weeks wherein the said composition reduces the parasitic load of spleen up to 81.6%.
- the said composition is preferably administered at a unit dose of 100 mg/Kg body weight at least for three weeks wherein the said composition reduces the parasitic load of liver up to 85.42%.
- the said composition further comprising the therapeutically effective amount of bioactive fraction and sodium antimony gluconate optionally along with one or more pharmaceutically acceptable carriers, additives and diluents wherein the ratio of bioactive fraction and sodium antimony gluconate is in the range of 200:1 to 20:1.
- the dosage of the said composition is administered at a unit dose of at least 150mg/Kg body weight of bioactive fraction and 5 mg/Kg body weight of sodium antimony gluconate to 200mg/Kg body weight of bioactive fraction and 10 mg/Kg body weight of sodium antimony gluconate.
- the dosage of the said composition is preferably administered at a unit dose of 150mg/Kg body weight of bioactive fraction and 5 mg/Kg body weight of sodium antimony gluconate at least for three weeks wherein the said composition reduces the parasitic load of spleen up to 95.1%.
- the dosage of the said composition is preferably administered at a unit dose of 150mg/Kg body weight of bioactive fraction and 5 mg/Kg body weight of sodium antimony gluconate at least for three weeks wherein the said composition reduces the parasitic load of liver up to 99.85%.
- the dosage of the said composition is preferably administered at a unit dose of 200mg/Kg body weight of bioactive fraction and 10 mg/Kg body weight of sodium antimony gluconate at least for three weeks wherein the said composition reduces the parasitic load of liver and spleen up to 100%.
- the said composition further comprising the therapeutically effective amount of bioactive fraction and piperine optionally along with one or more pharmaceutically acceptable carriers, additives and diluents wherein the ratio of bioactive fraction and piperine is in the range of 200:1 to 20:1.
- the dosage of the said composition is administered at a unit dose of lOOmg/Kg body weight of bioactive fraction and 0.5 mg/Kg body weight of piperine to 100 mg/Kg body weight of bioactive fraction and 5.0 mg/Kg body weight of piperine.
- the dosage of the said composition is preferably administered at a unit dose of 100mg/Kg body weight of bioactive fraction and 0.5 mg/Kg body weight of piperine at least for three weeks wherein the said composition reduces the parasitic load of spleen up to 98.8%.
- the dosage of the said composition is preferably administered at a unit dose of 100mg/Kg body weight of bioactive fraction and 0.5 mg/Kg body weight of piperine at least for three weeks wherein the said composition reduces the parasitic load of liver up to 79.8%.
- the dosage of the said composition is preferably administered at a unit dose of 100mg/Kg body weight of bioactive fraction and 5.0 mg/Kg body weight of piperine at least for three weeks wherein the said composition reduces the parasitic load of liver and spleen up to 100%.
- the said composition further comprising the therapeutically effective amount of bioactive fraction and sodium antimony gluconate and piperine optionally along with one or more pharmaceutically acceptable carriers, additives and diluents wherein the ratio of bioactive fraction and sodium antimony gluconate and piperine is in the range of 40: 1 : 0.2 to 40: 1 : 2
- the dosage of the said composition is administered at a unit dose of 100mg/Kg body weight of bioactive fraction and 2.5 mg/Kg body weight of sodium antimony gluconate and 0.5 mg/Kg body weight of piperine to 100mg/Kg body weight of bioactive fraction and 2.5 mg/Kg body weight of sodium antimony gluconate and 5.0 mg/Kg body weight of piperine.
- the dosage of the said composition is preferably administered at a unit dose of 100mg/Kg body weight of bioactive fraction and 2.5 mg/Kg body weight of sodium antimony gluconate and 5.0mg/Kg body weight of piperine at least for three weeks wherein the said composition reduces the parasitic load of liver and spleen up to 100%.
- the invention provides a method of treating visceral leishmaniasis in a subject, wherein the said method comprising the step of administering to the subject a pharmaceutical composition comprising the therapeutically effective amount of said bioactive fraction obtained from the leaves of the plant night jasmine or selected from the combination comprising the bioactive fraction and sodium antimony gluconate or bioactive fraction and piperine or bioactive fraction and sodium antimony gluconate , and piperine optionally along with one or more pharmaceutically acceptable carriers, additives, lubricant and diluents.
- the said subject is mammal including human.
- the additives used are selected from the group comprising of proteins, carbohydrates, sugar, talc, magnesium state, cellulose, calcium, carbohydrate, starch-gelatin paste.
- the said method further comprising the step of administering to the subject a pharmaceutical composition comprising therapeutically effective amount of bioactive fraction obtained from the leaves of the plant night jasmine optionally along with one or more pharmaceutically acceptable carriers, additives and diluents.
- the said composition is administered at a unit dose of at least 100-to 200-mg/Kg-body weight.
- the said composition is preferably administered at a unit dose of 100-mg/Kg body weight at least for three weeks wherein the said composition reduces the parasitic load of spleen up to 81.6%.
- the said composition is preferably administered at a unit dose of 100-mg/Kg body weight at least for three weeks wherein the said composition reduces the parasitic load of liver up to 85.42%.
- the said method further comprising the step of administering to the subject a pharmaceutical composition comprising the therapeutically effective amount of bioactive fraction and sodium antimony gluconate optionally along with one or more pharmaceutically acceptable carriers, additives and diluents wherein the ratio of bioactive fraction and sodium antimony gluconate is in the range of 200:1 to 20:1.
- the said composition is administered at a unit dose of at least 150mg/Kg body weight of bioactive fraction and 5 mg/Kg body weight of sodium antimony gluconate to 200mg/Kg body weight of bioactive fraction and 10 mg/Kg body weight of sodium antimony gluconate.
- the said composition is preferably administered at a unit dose of 150mg/Kg body weight of bioactive fraction and 5 mg/Kg body weight of sodium antimony gluconate at least for three weeks wherein the said composition reduces the parasitic load of spleen up to 95.1 %.
- the said composition is preferably administered at a unit dose of 150mg/Kg body weight of bioactive fraction and 5 mg/Kg body weight of sodium antimony gluconate at least for three weeks wherein the said composition reduces the parasitic load of liver up to 99.85%.
- the said composition is preferably administered at a unit dose of 200mg/Kg body weight of bioactive fraction and 10 mg/Kg body weight of sodium antimony gluconate at least for three weeks wherein the said composition reduces the parasitic load of liver and spleen up to 100%.
- the said wherein the said method further comprising the step of administering to the subject .
- a pharmaceutical composition comprising the therapeutically effective amount of bioactive fraction and piperine optionally along with one or more pharmaceutically acceptable carriers, additives and diluents wherein the ratio of bioactive fraction and piperine is in the range of200:l to 20:l.
- the said composition is administered at a unit dose of lOOmg/Kg body weight of bioactive fraction and 0.5 mg/Kg body weight of piperine to 100 mg/Kg body weight of bioactive fraction and 5.0 mg/Kg body weight of piperine.
- the said composition is preferably administered at a unit dose of lOOmg/Kg body weight of bioactive fraction and 0.5 mg/Kg body weight of piperine at least for three weeks wherein the said composition reduces the parasitic load of spleen up to 98.8%.
- the said composition is preferably administered at a unit dose of 100mg/Kg body weight of bioactive fraction and 0.5 mg/Kg body weight of piperine at least for three weeks wherein the said composition reduces the parasitic load of liver up to 79.8%.
- the said composition is preferably administered at a unit dose of 100mg/Kg body weight of bioactive fraction and 5.0 mg/Kg body weight of piperine at least for three weeks wherein the said composition reduces the parasitic load of liver and spleen up to 100%.
- the said wherein the said method further comprising the step of administering to the subject a pharmaceutical composition comprising the therapeutically effective amount of bioactive fraction and sodium antimony gluconate and piperine optionally along with one or more pharmaceutically acceptable carriers, additives and diluents wherein the ratio of bioactive fraction and sodium antimony gluconate and piperine is in the range of 40: 1: 0.2 to 40: 1: 2.
- the said composition is administered at a unit dose of 100mg/Kg body weight of bioactive fraction and 2.5 mg/Kg body weight of sodium antimony gluconate and 0.5 mg/Kg body weight of piperine to 100mg/Kg body weight of bioactive fraction and 2.5 mg/Kg body weight of sodium antimony gluconate and 5.0 mg/Kg body weight of piperine.
- the said composition is preferably administered at a unit dose of 100mg/Kg body weight of bioactive fraction and 2.5 mg/Kg body weight of sodium antimony gluconate and 5.0mg/Kg body weight of piperine at least for three weeks wherein the said composition reduces the parasitic load of liver and spleen up to 100%.
- the administration route used is selected from the group consisting of oral, intraperitoneal, intravenous, intramuscular.
- the said form for oral route is selected from the group consisting of capsule, syrup, concentrate, powder and granules.
- the effective dose of the said composition is administered in such a manner that it does not interfere the other physiological activities.
- the effective dose of the said composition is administered in such a manner that it does not create any clinical and pathological toxicity to the subject.
- the present invention also provides a method for the isolation of compound calceolarioside A from the bioactive fraction, wherein the said method comprising the steps of: a) extracting the powdered dry mass of fresh leaves of Nyctanthes arbortristis with alcohol, b) concentrating the extract obtained from step (a) under reduced pressure at atleast 40 °C to obtain semi solid mass; c) performing repeatedly chromatography using organic solvent of the said mass as obtained from step (b) to get bioactive fraction; d) performing thin layer chromatography to get desired compound calceolarioside A.
- the leaves of Nyctanthes arbortristis are collected from Jhargram, West Bengal and voucher specimen submitted to Botanical Survey of India, Shibpur, Howrah ( vide voucher specimen no. NAlOl, dated 28.06.2006).
- the alcohol used is selected from the group comprising of methanol, ethanol, n-butanol.
- the semi solid mass of step (b) is chromatographed over a silica gel column having mesh size 60nm-120nm.
- the organic solvent used is chloroform.
- the present invention is related to a method for novel drug development from natural sources without any clinical and pathological toxic effects, wherein the said method comprising preparing methanolic extract of night jasmine leaf, chromatographic purification of said extract, isolation bioactive fraction of containing a compound calceolarioside A , treatment of L.
- a method for using night jasmine leaf extract as leishmanicidal agent comprising a) methanolic extraction of night jasmine leaf and chromatographic purification to isolate calceolarioside A containing bioactive fraction (PE). b) administering bioactive fraction at least 100-200mg/kg body weight/day through i.p for a period of at least three weeks to the L. donovani infected chronic golden hamster model. c) administering bioactive fraction at least 100-200mg/kg body weight/day and SAG at least 5 and 10mg/kg body weight/day through i .p for a period of at least three weeks to the L. donovani infected chronic golden hamster model. d) antileishmanial activity of calceolarioside A against axenic L. donovani promastigote culture.
- a pharmaceutical composition of the present invention is useful as leishmanicidal agent against chronic visceral leishmaniasis.
- SAG Sodium Antimony Gluconate
- PE Calceolariside A containing bioactive fraction
- the pure compound calceolarioside A was collected by preparative thin layer chromatography from this semi-pure bioactive fraction of example 2. Identification of the pure compound was done with 1 H, and 13 C Nuclear Magnetic Resonance (NMR). Spectra recorded with a Bruker DPX-300 machine. Both positive and negative ion- mode mass spectrometry were done with Micromass Q-Tof microTM to determine the molecular weight.
- Leishmania donovani Ag83 was isolated from an Indian kala-azar patient and maintained in Golden hamster (Mesocrisetm auratus) model (A.K Ghosh, F. K. Bhattacharya and D.K Ghosh, Leishmania donovani: amastigote inhibition and mode of action of berberine (1985) Experimental Parasitology, Vol. 60 pp 404-413). Liquid parasite culture was done in Ml 99 media (Sigma, USA) supplemented with 10% Fetal Bovine Serum (Sigma, USA) at 22°C culture room. Stationary phase promastigotes of L .donovani were used for in- vitro assay.
- the in-vitro activity of crude extract, bioactive fraction and pure compound calceolarioside A was determined on stationary phase promastigotes of L. donovani Ag83 strain.
- the stationary phase promastigotes were collected from parasite culture by centrifugation and re-suspended with 10% FBS supplemented Ml 99 medium.
- For drug sensitivity assay 50mg/ml stock solution for each test material were prepared with minimum amount of DMSO and PBS and serially diluted with sterile PBS to achieve the desired dose. Assay was performed in sterile tubes with 450 ⁇ l of parasite suspension. Then 50 ⁇ l of test solutions were added to each tube and one control only with solvent (DMSO and PBS) was maintained. Tubes were incubated at 22 0 C for 72hrs and data was collected after each 24hrs .Mortality rate of the parasite was determined microscopically on Neubauer Counter Chamber (HBG, Germany).
- HPLC Shiadzu, Japan chromatogram of bioactive fraction containing calceolarioside A is obtained from methanolic crude extract of night jasmine leaf. The analysis was performed with C 18 column (250x4 mm I. D; SGE International PTY, Australia) attached with a Guard column (Supelco) a Rheodyne injection valve with a 100- ⁇ l loop and PDA detector (SPD-M 10 Avp).
- a linear gradient elution was carried out with methanol: water mixtures from 0:100,(v/v) to 100:0,(v/v) within 0 to 60 min at a flow rate of 0.5 ml/min.
- the column was maintained at room temperature and the detection was performed at UV 210 nm and 254 nm.
Landscapes
- Health & Medical Sciences (AREA)
- Life Sciences & Earth Sciences (AREA)
- Veterinary Medicine (AREA)
- Chemical & Material Sciences (AREA)
- Medicinal Chemistry (AREA)
- Pharmacology & Pharmacy (AREA)
- Animal Behavior & Ethology (AREA)
- General Health & Medical Sciences (AREA)
- Public Health (AREA)
- Epidemiology (AREA)
- Natural Medicines & Medicinal Plants (AREA)
- Molecular Biology (AREA)
- General Chemical & Material Sciences (AREA)
- Biotechnology (AREA)
- Organic Chemistry (AREA)
- Chemical Kinetics & Catalysis (AREA)
- Tropical Medicine & Parasitology (AREA)
- Engineering & Computer Science (AREA)
- Alternative & Traditional Medicine (AREA)
- Nuclear Medicine, Radiotherapy & Molecular Imaging (AREA)
- Botany (AREA)
- Medical Informatics (AREA)
- Microbiology (AREA)
- Mycology (AREA)
- Medicines Containing Plant Substances (AREA)
- Pharmaceuticals Containing Other Organic And Inorganic Compounds (AREA)
- Medicines Containing Material From Animals Or Micro-Organisms (AREA)
Abstract
This invention relates to the leishmanicidal activity of night jasmine leaf extract, wherein the said method comprises the steps of, preparing the methanolic extract of night jasmine leaf, chromatographic purification of said extract to obtain bioactive fraction containing calceolarioside A, isolation and characterization of calceolarioside A, leishmanicidal activity of calceolarioside A against axenic promastigote culture of Leishmania donovani, treatment of L. donovani infected chronic golden hamster with bioactive fraction for a period of 21 days for detection of leishmanicidal activity in vivo, administering of PE in normal hamster to obtain non-toxic nature, combination therapy of bioactive fraction + SAG against said model for synergistic potentiation of antileishmanial activity and complete clearance of parasitemia from liver and spleen as reflected by LDU wherein comprising a potential medicament against kala-azar.
Description
A PHARMACEUTICAL COMPOSITION USEFUL AS A LEISHMANICIDAL
AGENT
Field of the invention: The present invention relates to a bioactive fraction obtained from the leaves of the plant night jasmine.
More particularly, it relates to the use of said bioactive fraction in the treatment of visceral leshmaniasis.
The present invention also relates to the use of the compound calceolarioside A as a leishmanicidal agent.
It also relates to a pharmaceutical composition useful as a leishmanicidal agent, wherein the said composition comprising the therapeutically effective amount of said bioactive fraction obtained from the leaves of the plant night jasmine or selected from the combination comprising the bioactive fraction and sodium antimony gluconate or bioactive fraction and piperine or bioactive fraction and sodium antimony gluconate and piperine optionally along with one or more pharmaceutically acceptable carriers, additives, lubricant and diluents.
Further, it relates to a method of treating visceral leishmaniasis in a subject using the said pharmaceutical composition. The present invention also relates to a method for the isolation of compound calceolarioside A from the bioactive fraction obtained from the leaves of the plant
Nyctanthes arbortristis.
Background and prior art of the invention: Infection with the protozoan parasite Leishmania donovani is a major health problem with significant morbidity and mortality. Leishmaniasis is estimated to afflict 12 million people worldwide, causing disease ranging from skin lesion in cutaneous leishmaniasis to a progressive and fatal hepatosplenomegaly in visceral leishmaniasis (CR Davies, P. Kaye, S.L.Croft and S. Sundar, Leishmaniasis: new approaches to disease control, British Medicinal Journal. (2003) Vol. 15 pp. 377-382). Chemotherapy is still a major challenge to combat leishmaniasis despite the rapid and tremendous development of medicinal and pharmaceutical industry in recent decades. Even today, the pentavalent antimonials sodium antimony gluconate (SAG) has remained the first-
C0NF1RMAT10N COPY
line treatment of visceral leishmaniasis (VL) or kala-azar with its extended treatment regimens, peritoneal administration and toxic side effects (Santu Bandyopadhyay, Bikash Pal, Samir Bhattacharya, Mitali Ray, Keshab Chandra Roy (2003) U.S.Patent no. 6,610,332). Non-availability of antileishmanial vaccine makes the situation more difficult. Recently, co-infection of visceral leishmaniasis patients with HIV makes the search for new agents for the treatment of leishmaniasis more urgent. Compounding this problem is the fact that many countries and regions where the disease is endemic are economically poor. This lack of an effective antileishmanial drug and urgency of the present drug situation has generated a renewed interest in the study of traditional remedies to develop new antileishmanial chemotherapeutic agents with better activity and less toxicity. Few medicinal plants have been studied for the treatment of this parasitic disease but their value as drug candidates cannot be fully assessed since systemic in vivo studies on animal models were not generated. In addition, extensive use of herbal therapy in leishmania endemic regions has created interest in evaluation of plant remedies used in traditional medicine as sources of potential antileishmanial agent (M.M. Iwu, J.E. Jackson and B. G. Schuster, Medicinal Plants in the Fight against Leishmaniasis, Parasitology Today, (1994) Vol.lO.no.2, pp.65-68). Nyctanthes arbortristis, is a traditionally used medicinal plant of India commonly used as an anthelmintic, antibilious, diaphoretic etc. and fpr the treatment of various diseases such as fever, rheumatism, intestinal worm disinfections etc. (The Wealth of India, Raw Materials, (1966) Vol. 7, pp 69-70). Tribal people of central India used various parts of this plant for relieving cough, dysentery, snakebite, sores etc. (S.KJain, Dictionary of Indian folk medicine and Ethnobotany, 1991). Most of the recent bioactivity studies on N. arbortristis has been carried out from seed extract and iridoid glycosides (arbortristoside A, B, C, D, E) were isolated which showed antiviral (Anita Rathore, Vandita Srivastava, K.C.Srivastava and J.S. Tandon, Phytochemistry (1990) Vol.29 pp.1917-1920) or antileishmanial activity (J.S.Tandon, Vandita Srivastava and P.Y.Guru, Journal of Natural Products, (1991) Vol.54 pp.l 102-1104). Wherein, in the present study calceolarioside A, a new class of compound to be reported from this plant, was isolated and purified from the methanolic extract of night jasmine leaf and showed significant antileishmanial activity both in vitro and in vivo test systems. This
is the first isolation report of calceolarioside A from this plant as well as the first antileishmanial activity report of calceolarioside A.
Objects of the invention: The main object of the present invention is to provide a bioactive fraction obtained from the leaves of the plant night jasmine.
Another object of the present invention is to provide the use of said bioactive fraction in the treatment of visceral leishmaniasis. Further object of the present invention is to provide the use of the compound calceolarioside A as a leishmanicidal agent.
Yet another object of the present invention is to provide a pharmaceutical composition useful as a leishmanicidal agent, wherein the said composition comprising the therapeutically effective amount of said bioactive fraction obtained from the leaves of the plant night jasmine or selected from the combination comprising the bioactive fraction and sodium antimony gluconate or bioactive fraction and piperine or bioactive fraction and sodium antimony gluconate and piperine optionally along with one or more pharmaceutically acceptable carriers, additives, lubricant and diluents. Still another object of the present invention is to provide a method for the isolation of compound calceolarioside A from the bioactive fraction obtained from the leaves of the plant Nyctanthes arbortristis.
Summary of the invention:
The present invention deals with a bioactive fraction obtained from the leaves of the plant night jasmine comprising a) calceolarioside A b) unidentified compounds c) kaempferol glycoside. It also provides the use of the said fraction and the compound calceolarioside A in the treatment of visceral leishmaniasis. Further, it also relates to a pharmaceutical composition and method of treating visceral leishmaniasis using said composition. The invention also relates to a method of isolation of said compound from the bioactive fraction obtained from the leaves of the plant Nyctanthes arbortristis.
Brief description of the figures:
Figure 1 represents the effect of crude methanolic extract of Nyctanthes arbortristis leaf and PE on the growth of Leishmania donovani promastigotes in vitro in time and dose
dependent manner. Viable cells, i.e., promastigotes (xlO6)/nil of culture. Data are shown for one representative experiment out of three with similar results. Solvent was DMSO+PBS.
Figure 2 represents the effect of Nyctanthes arbortristis crude methanolic leaf extract against established infection of visceral leishmaniasis in golden hamster model. Two weeks infected golden hamsters were treated with crude extract at the dose of lOmg/kg, 20mg/kg, 40 mg/kg and 60mg/kg body weight for 21 days. Controls were infected untreated animals. Crude extract was administered through i.p route. Treated animals received total six shots of crude extract during three weeks of treatment schedule. Animals were sacrificed after six weeks of infection. Levels of parasite burden in spleen and liver are expressed in Leishman-Donovan units (LDU). Figure 3 represents the effect of methanolic extract of Nyctanthes arhortritis leaf and SAG in established infection of Leishmania donovani. Four weeks infected golden hamsters were treated intra-peritoneally with crude methanolic extract at the doses of 10mg/kg body weight, 20 mg/kg body weight and SAG 10mg/kg body weight for 7 days respectively. Controls were infected untreated animals. Treated animals received one shot in one week to complete the treatment. Level of parasite burden in spleen is expressed in Leishman-Donovan units (LDU). DMSO: PBS=DMSO+PBS as solvent of crude extract Figure 4 represents the effect of bioactive fraction and combined therapy of bioactive fraction + SAG in established infection of visceral leishmaniasis. Two weeks infected golden hamsters were treated intra-peritoneally with bioactive fraction at the dose of lOOmg/kg body weight, SAG at dose of 5mg/kg body weight and bioactive fraction +SAG at the dose of 100+5 mg/kg body weight for 21 days. Controls were infected untreated animals. Treated animals received total three shots during three weeks of treatment schedule. Animals were sacrificed after six weeks of infection. Level of parasite burden in spleen and liver are expressed in Leishman-Donovan units (LDU). Figure 5 represents the therapeutic efficacy of bioactive fraction and combined therapy of bioactive fraction + SAG in chronic visceral leishmaniasis. Eight weeks infected golden hamsters were treated intra-peritoneally with bioactive fraction at the doses of 100,150,200mg/kg body weight and bioactive fraction +SAG at the doses of 150+5,200+5,200+10mg/kg body weight respectively for 21 days. Controls were
infected untreated animals. Treated animals received total six shots during three weeks of treatment schedule. Animals were sacrificed after 12 weeks of infection. Level of parasite burden in spleen and liver are expressed in Leishman-Donovan units (LDU). Figure 6 represents the effect of calceolarioside A on the growth of Leishmania donovani promastigotes in vitro in time and dose dependent manner. Viable cells, i.e., promastigotes (xlO6)/ml of culture. Data are shown for one representative experiment out of three with similar results. NAl=CalceolariosideA; Solvent=DMSO+PBS Figure 7 represents the effect of bioactive fraction and combined therapy of bioactive fraction, SAG and piperine against established infection of visceral leishmaniasis in golden hamster model. Effect of bioactive fraction, combined therapy of bioactive fraction + piperine, bioactive fraction + SAG and bioactive fraction + SAG + piperine against established infection of visceral leishmaniasis in golden hamster model. Two weeks infected golden hamsters were treated with bioactive fraction at the dose of lOOmg/kg body weight, bioactive fraction + piperine at the dose of 100 + 5 mg/kg body weight and bioactive fraction + SAG at the dose of 100 + 5mg/kg body weight and bioactive fraction + SAG + piperine at the dose of 100mg+2.5mg+5mg/kg body weight were given for 21 days. Controls were infected untreated animals. Bioactive fraction and SAG were administered i.p. and free piperine was administered orally as dry powder. Treated animals received total six shots of bioactive fraction and SAG (i.p.) and free piperine was administered orally as dry powder before two hours of each bioactive fraction dose during three weeks of treatment schedule. Animals were sacrificed after six weeks of infection. Levels of parasite burden in spleen and liver are expressed in Leishman-Donovan units (LDU). Fig 8: HPLC chromatogram of bioactive fraction obtained from methanolic extract of night jasmine leaf.
First Peak: Calceolarioside A
Second Peak: Kaempferol glycoside
Two unidentified very small peak
Fig 9: HPLC chromatogram of calceolarioside A obtained from methanolic extract of night jasmine leaf.
Detailed description of the invention:
Accordingly, the present invention provides a bioactive fraction obtained from the leaves of the plant night jasmine, wherein the said fraction comprising a) calceolarioside A 39.62% b) kaempferol glycoside 34.8%, c) two unidentified compound 5.2% & 5.8% and d) unidentified unresolved fraction 14.6%, wherein said weight percentages are percentages in the total weight of the fraction.
In an embodiment of the present invention, the said fraction is useful as a leishmanicidal agent.
In another embodiment of the present invention, the said fraction is non toxic to liver and kidney at the dose of 200mg/Kg body weight / day at least for three weeks.
The present invention also provides the use of the bioactive fraction in the treatment of visceral leshmaniasis.
Further, it provides the use of the compound calceolarioside A as a leishmanicidal agent. In an embodiment of the present invention, the said compound reduces the viability of
L. donovani promastigotes in vitro uptolOO % at the dose of 80 μg/ml of culture media for 24 hrs.
The present invention also provides a pharmaceutical composition useful as a leishmanicidal agent, wherein the said composition comprising the therapeutically effective amount of said bioactive fraction obtained from the leaves of the plant night jasmine or selected from the combination comprising the bioactive fraction and sodium antimony gluconate or bioactive fraction and piperine or bioactive fraction and sodium antimony gluconate and piperine optionally along with one or more pharmaceutically acceptable carriers, additives, lubricant and diluents. In an embodiment of the present invention, the additives used are selected from the group comprising of proteins, carbohydrates, sugar, talc, magnesium state, cellulose, calcium, carbohydrate, starch-gelatin paste.
In another embodiment of the present invention, the said composition further comprising the therapeutically effective amount of bioactive fraction obtained from the leaves of the plant night jasmine optionally along with one or more pharmaceutically acceptable carriers, additives and diluents.
W
7
Further, in another embodiment of the present invention, the dosage of the said composition is administered at a unit dose of at least 100 to 200 mg/Kg body weight. In yet another embodiment of the present invention, the said composition is preferably administered at a unit dose of 100 mg/Kg body weight at least for three weeks wherein the said composition reduces the parasitic load of spleen up to 81.6%.
In still another embodiment of the present invention, the said composition is preferably administered at a unit dose of 100 mg/Kg body weight at least for three weeks wherein the said composition reduces the parasitic load of liver up to 85.42%. In still another embodiment of the present invention, the said composition further comprising the therapeutically effective amount of bioactive fraction and sodium antimony gluconate optionally along with one or more pharmaceutically acceptable carriers, additives and diluents wherein the ratio of bioactive fraction and sodium antimony gluconate is in the range of 200:1 to 20:1. In still another embodiment of the present invention, the dosage of the said composition is administered at a unit dose of at least 150mg/Kg body weight of bioactive fraction and 5 mg/Kg body weight of sodium antimony gluconate to 200mg/Kg body weight of bioactive fraction and 10 mg/Kg body weight of sodium antimony gluconate. In still another embodiment of the present invention, wherein the dosage of the said composition is preferably administered at a unit dose of 150mg/Kg body weight of bioactive fraction and 5 mg/Kg body weight of sodium antimony gluconate at least for three weeks wherein the said composition reduces the parasitic load of spleen up to 95.1%.
In still another embodiment of the present invention, wherein the dosage of the said composition is preferably administered at a unit dose of 150mg/Kg body weight of bioactive fraction and 5 mg/Kg body weight of sodium antimony gluconate at least for three weeks wherein the said composition reduces the parasitic load of liver up to 99.85%.
In still another embodiment of the present invention, wherein the dosage of the said composition is preferably administered at a unit dose of 200mg/Kg body weight of bioactive fraction and 10 mg/Kg body weight of sodium antimony gluconate at least for three weeks wherein the said composition reduces the parasitic load of liver and spleen up to 100%.
In still another embodiment of the present invention, wherein the said composition further comprising the therapeutically effective amount of bioactive fraction and piperine optionally along with one or more pharmaceutically acceptable carriers, additives and diluents wherein the ratio of bioactive fraction and piperine is in the range of 200:1 to 20:1.
In still another embodiment of the present invention, wherein the dosage of the said composition is administered at a unit dose of lOOmg/Kg body weight of bioactive fraction and 0.5 mg/Kg body weight of piperine to 100 mg/Kg body weight of bioactive fraction and 5.0 mg/Kg body weight of piperine. In still another embodiment of the present invention, wherein the dosage of the said composition is preferably administered at a unit dose of 100mg/Kg body weight of bioactive fraction and 0.5 mg/Kg body weight of piperine at least for three weeks wherein the said composition reduces the parasitic load of spleen up to 98.8%. In still another embodiment of the present invention, wherein the dosage of the said composition is preferably administered at a unit dose of 100mg/Kg body weight of bioactive fraction and 0.5 mg/Kg body weight of piperine at least for three weeks wherein the said composition reduces the parasitic load of liver up to 79.8%. In still another embodiment of the present invention, the dosage of the said composition is preferably administered at a unit dose of 100mg/Kg body weight of bioactive fraction and 5.0 mg/Kg body weight of piperine at least for three weeks wherein the said composition reduces the parasitic load of liver and spleen up to 100%. In still another embodiment of the present invention, the said composition further comprising the therapeutically effective amount of bioactive fraction and sodium antimony gluconate and piperine optionally along with one or more pharmaceutically acceptable carriers, additives and diluents wherein the ratio of bioactive fraction and sodium antimony gluconate and piperine is in the range of 40: 1 : 0.2 to 40: 1 : 2 In still another embodiment of the present invention, the dosage of the said composition is administered at a unit dose of 100mg/Kg body weight of bioactive fraction and 2.5 mg/Kg body weight of sodium antimony gluconate and 0.5 mg/Kg body weight of piperine to 100mg/Kg body weight of bioactive fraction and 2.5 mg/Kg body weight of sodium antimony gluconate and 5.0 mg/Kg body weight of piperine.
In still another embodiment of the present invention, the dosage of the said composition is preferably administered at a unit dose of 100mg/Kg body weight of bioactive fraction and 2.5 mg/Kg body weight of sodium antimony gluconate and 5.0mg/Kg body weight of piperine at least for three weeks wherein the said composition reduces the parasitic load of liver and spleen up to 100%.
Further, the invention provides a method of treating visceral leishmaniasis in a subject, wherein the said method comprising the step of administering to the subject a pharmaceutical composition comprising the therapeutically effective amount of said bioactive fraction obtained from the leaves of the plant night jasmine or selected from the combination comprising the bioactive fraction and sodium antimony gluconate or bioactive fraction and piperine or bioactive fraction and sodium antimony gluconate , and piperine optionally along with one or more pharmaceutically acceptable carriers, additives, lubricant and diluents. In an embodiment of the present invention, the said subject is mammal including human.
In another embodiment of the present invention, the additives used are selected from the group comprising of proteins, carbohydrates, sugar, talc, magnesium state, cellulose, calcium, carbohydrate, starch-gelatin paste. In another embodiment of the present invention, the said method further comprising the step of administering to the subject a pharmaceutical composition comprising therapeutically effective amount of bioactive fraction obtained from the leaves of the plant night jasmine optionally along with one or more pharmaceutically acceptable carriers, additives and diluents. Further, in an embodiment of the present embodiment of the present invention, the said composition is administered at a unit dose of at least 100-to 200-mg/Kg-body weight. In yet another embodiment of the present invention, the said composition is preferably administered at a unit dose of 100-mg/Kg body weight at least for three weeks wherein the said composition reduces the parasitic load of spleen up to 81.6%. In still another embodiment of the present invention, the said composition is preferably administered at a unit dose of 100-mg/Kg body weight at least for three weeks wherein the said composition reduces the parasitic load of liver up to 85.42%.
In still another embodiment of the present invention, the said method further comprising the step of administering to the subject a pharmaceutical composition comprising the therapeutically effective amount of bioactive fraction and sodium antimony gluconate optionally along with one or more pharmaceutically acceptable carriers, additives and diluents wherein the ratio of bioactive fraction and sodium antimony gluconate is in the range of 200:1 to 20:1.
In still another embodiment of the present invention, the said composition is administered at a unit dose of at least 150mg/Kg body weight of bioactive fraction and 5 mg/Kg body weight of sodium antimony gluconate to 200mg/Kg body weight of bioactive fraction and 10 mg/Kg body weight of sodium antimony gluconate.
In still another embodiment of the present invention, the said composition is preferably administered at a unit dose of 150mg/Kg body weight of bioactive fraction and 5 mg/Kg body weight of sodium antimony gluconate at least for three weeks wherein the said composition reduces the parasitic load of spleen up to 95.1 %. In still another embodiment of the present invention, the said composition is preferably administered at a unit dose of 150mg/Kg body weight of bioactive fraction and 5 mg/Kg body weight of sodium antimony gluconate at least for three weeks wherein the said composition reduces the parasitic load of liver up to 99.85%. In still another embodiment of the present invention, the said composition is preferably administered at a unit dose of 200mg/Kg body weight of bioactive fraction and 10 mg/Kg body weight of sodium antimony gluconate at least for three weeks wherein the said composition reduces the parasitic load of liver and spleen up to 100%. In still another embodiment of the present invention, the said wherein the said method further comprising the step of administering to the subject . a pharmaceutical composition comprising the therapeutically effective amount of bioactive fraction and piperine optionally along with one or more pharmaceutically acceptable carriers, additives and diluents wherein the ratio of bioactive fraction and piperine is in the range of200:l to 20:l. In still another embodiment of the present invention, the said composition is administered at a unit dose of lOOmg/Kg body weight of bioactive fraction and 0.5 mg/Kg body weight of piperine to 100 mg/Kg body weight of bioactive fraction and 5.0 mg/Kg body weight of piperine.
In still another embodiment of the present invention, the said composition is preferably administered at a unit dose of lOOmg/Kg body weight of bioactive fraction and 0.5 mg/Kg body weight of piperine at least for three weeks wherein the said composition reduces the parasitic load of spleen up to 98.8%. In still another embodiment of the present invention, the said composition is preferably administered at a unit dose of 100mg/Kg body weight of bioactive fraction and 0.5 mg/Kg body weight of piperine at least for three weeks wherein the said composition reduces the parasitic load of liver up to 79.8%. In still another embodiment of the present invention, the said composition is preferably administered at a unit dose of 100mg/Kg body weight of bioactive fraction and 5.0 mg/Kg body weight of piperine at least for three weeks wherein the said composition reduces the parasitic load of liver and spleen up to 100%.
In still another embodiment of the present invention, the said wherein the said method further comprising the step of administering to the subject a pharmaceutical composition comprising the therapeutically effective amount of bioactive fraction and sodium antimony gluconate and piperine optionally along with one or more pharmaceutically acceptable carriers, additives and diluents wherein the ratio of bioactive fraction and sodium antimony gluconate and piperine is in the range of 40: 1: 0.2 to 40: 1: 2. In still another embodiment of the present invention, the said composition is administered at a unit dose of 100mg/Kg body weight of bioactive fraction and 2.5 mg/Kg body weight of sodium antimony gluconate and 0.5 mg/Kg body weight of piperine to 100mg/Kg body weight of bioactive fraction and 2.5 mg/Kg body weight of sodium antimony gluconate and 5.0 mg/Kg body weight of piperine. In still another embodiment of the present invention, the said composition is preferably administered at a unit dose of 100mg/Kg body weight of bioactive fraction and 2.5 mg/Kg body weight of sodium antimony gluconate and 5.0mg/Kg body weight of piperine at least for three weeks wherein the said composition reduces the parasitic load of liver and spleen up to 100%. In still another embodiment of the present invention, the administration route used is selected from the group consisting of oral, intraperitoneal, intravenous, intramuscular.
In still another embodiment of the present invention, the said form for oral route is selected from the group consisting of capsule, syrup, concentrate, powder and granules. In still another embodiment of the present invention, the effective dose of the said composition is administered in such a manner that it does not interfere the other physiological activities.
In still another embodiment of the present invention, the effective dose of the said composition is administered in such a manner that it does not create any clinical and pathological toxicity to the subject.
Still the present invention also provides a method for the isolation of compound calceolarioside A from the bioactive fraction, wherein the said method comprising the steps of: a) extracting the powdered dry mass of fresh leaves of Nyctanthes arbortristis with alcohol, b) concentrating the extract obtained from step (a) under reduced pressure at atleast 40 °C to obtain semi solid mass; c) performing repeatedly chromatography using organic solvent of the said mass as obtained from step (b) to get bioactive fraction; d) performing thin layer chromatography to get desired compound calceolarioside A. In an embodiment of the present invention, the leaves of Nyctanthes arbortristis are collected from Jhargram, West Bengal and voucher specimen submitted to Botanical Survey of India, Shibpur, Howrah ( vide voucher specimen no. NAlOl, dated 28.06.2006). In another embodiment of the present invention, the alcohol used is selected from the group comprising of methanol, ethanol, n-butanol.
Further, in an embodiment of the present invention, the semi solid mass of step (b) is chromatographed over a silica gel column having mesh size 60nm-120nm.
In yet another embodiment of the present invention, the organic solvent used is chloroform. The present invention is related to a method for novel drug development from natural sources without any clinical and pathological toxic effects, wherein the said method comprising preparing methanolic extract of night jasmine leaf, chromatographic
purification of said extract, isolation bioactive fraction of containing a compound calceolarioside A , treatment of L. donovani infected chronic golden hamster model by administering (i.p) bioactive fraction at lOOmg, 150mg and 200mg/kg body weight/day for three weeks, determination of LDU for the detection of parasite load in liver, spleen and bone marrow, combination therapy treatment of bioactive fraction + sodium antimony gluconate at a dose of 150mg + 5mg and 200mg + 10mg/kg body weight/day respectively for three weeks to obtain complete clearance of parasitemia, testing the leishmanicidal activity of calceolarioside A detected against axenic culture of L. donovani promastigotes, and administering bioactive fraction in normal healthy animal to obtain clinical and pathological non-toxic nature. -
A method for using night jasmine leaf extract as leishmanicidal agent wherein the said method comprising a) methanolic extraction of night jasmine leaf and chromatographic purification to isolate calceolarioside A containing bioactive fraction (PE). b) administering bioactive fraction at least 100-200mg/kg body weight/day through i.p for a period of at least three weeks to the L. donovani infected chronic golden hamster model. c) administering bioactive fraction at least 100-200mg/kg body weight/day and SAG at least 5 and 10mg/kg body weight/day through i .p for a period of at least three weeks to the L. donovani infected chronic golden hamster model. d) antileishmanial activity of calceolarioside A against axenic L. donovani promastigote culture.
A pharmaceutical composition of the present invention is useful as leishmanicidal agent against chronic visceral leishmaniasis. Abbreviations used:
SAG=Sodium Antimony Gluconate
LDU=Leishman-Donovan Unit
PE =Calceolariside A containing bioactive fraction
The following examples are given by way of illustration of the present invention and should not be construed to limit the scope of present invention
EXAMPLE 1
8 KG of fresh leaves of Nyctanthes arbortristis was thoroughly washed in sterile water, shed dried and powdered, then extracted overnight with 8 L of methanol at room temperature. The process was repeated thrice. The total pool of crude extract was filtered through Whatman filter paper No 1 and the filtrate was collected. The combined filtrate was evaporated to dryness under reduced pressure below 40° C and kept at room temperature in vacuum desiccator under high vacuum yielding a semisolid mass weighing 1360gm. Successive bio-activity guided fractionation and purification were performed with this dried extract.
EXAMPLE 2
The crude extract was triturated with Ethyl Acetate (GR Merck) and the rest was again triturated with n-Butanol (GR Merck). The n-Butanol fraction was evaporated to dryness under reduced pressure at 40°C.This extract was chromatographed over a silica gel column (mesh size 60-120) beginning the elution with Chloroform (GR Merck). Successive fractions were eluted from the column with increasing percentages of methanol in chloroform. When the fractions with 8-10% methanol were evaporated, a slightly brown colored amorphous powder was collected as a semi-pure bioactive fraction. All the in-vivo experiments were carried out with this bioactive fraction.
EXAMPLE 3
The pure compound calceolarioside A was collected by preparative thin layer chromatography from this semi-pure bioactive fraction of example 2. Identification of the pure compound was done with 1H, and 13C Nuclear Magnetic Resonance (NMR). Spectra recorded with a Bruker DPX-300 machine. Both positive and negative ion- mode mass spectrometry were done with Micromass Q-Tof micro™ to determine the molecular weight. IH NMR in CD3 ODO OQMHz) δ 7.61(lH,d,J=15.9Hz),7.07(lH,d,J=1.3Hz),6.98(lH,br d, J=8.1Hz),6.79(lH,d,J=8.1Hz), 6.69(2H, overlapping signal), 6.59(1H, br d, J=8.1 Hz), 6.31(1H, d, J=I 5.9Hz), 4.38(1H, d, J=7.8Hz), 4.07(1H, dt, J=8.7, 7.8Hz), 3.4-3.8 (several protons), 2.82(2H, T, J=7Hz), solvent signals at 4.9, 3.32 and 2.17 Hz.
13CNMR in CDSODfTSMHz) δ 167.6(C), 148.7(C), 146.6(C), 145.8(C), 145.1(C), 143.7(C), 130.5(C), 126.7(C), 122.1(CH), 120.3(CH), 116.1(CH), 115.5(CH), 115.3(CH), 114.3(CH), 113.8(CH), 103.4(CH), 75.1(CH), 74.8(CH), 74.3(CH), 71.5(CH), 71.2(CH2), 61.5 (CH2), 35.6 (CH2).
ESI MSφositive mode) Here, m/z=501(M+Na)+ ESI MSCnegative mode) Here, m/z=477(M-H)"
Yield of Methanolic crude extract from Dry leaf =17.48%
Yield of n-Butanol fraction from Dry leaf =14.96%
Yield of calceolarioside A containing Bioactive fraction (PE) from Dry leaf =3.2% Yield of calceolarioside A from Dry leaf =1.237% Yield of kaempferol glycoside from Dry leaf =1.088% Yield of calceolarioside A from PE =39.62% Yield of kaempferol glycoside from PE =34.8% Yield of Other unidentified compounds from PE =5.2% & 5.8%
EXAMPLE 4
Leishmania donovani Ag83 was isolated from an Indian kala-azar patient and maintained in Golden hamster (Mesocrisetm auratus) model (A.K Ghosh, F. K. Bhattacharya and D.K Ghosh, Leishmania donovani: amastigote inhibition and mode of action of berberine (1985) Experimental Parasitology, Vol. 60 pp 404-413). Liquid parasite culture was done in Ml 99 media (Sigma, USA) supplemented with 10% Fetal Bovine Serum (Sigma, USA) at 22°C culture room. Stationary phase promastigotes of L .donovani were used for in- vitro assay. The in-vitro activity of crude extract, bioactive fraction and pure compound calceolarioside A was determined on stationary phase promastigotes of L. donovani Ag83 strain. The stationary phase promastigotes were collected from parasite culture by centrifugation and re-suspended with 10% FBS supplemented Ml 99 medium. For drug sensitivity assay 50mg/ml stock solution for each test material were prepared with minimum amount of DMSO and PBS and serially diluted with sterile PBS to achieve the desired dose. Assay was performed in sterile
tubes with 450μl of parasite suspension. Then 50μl of test solutions were added to each tube and one control only with solvent (DMSO and PBS) was maintained. Tubes were incubated at 220C for 72hrs and data was collected after each 24hrs .Mortality rate of the parasite was determined microscopically on Neubauer Counter Chamber (HBG, Germany).
EXAMPLE 5
In-vivo antileishmanial activity of bioactive fraction in the L. donovani infected golden hamster model: Male 4-6 weeks old Golden hamster (M auratus) were infected with 2.5x107 amastigotes of L. donovani intracardially on day 0 and randomly allocated into groups of 6 animals each. The amastigotes were obtained from the spleen of a two months infected donor hamster for infection. The bioactive fraction was dissolved in minimum amount of DMSO and then diluted accordingly with sterile PBS. SAG was diluted in sterile PBS. The formulations were administered, once or twice per week for one or three successive weeks, intraperitonially at different doses after two weeks or four weeks or eight weeks of infection according to the experiment. Fresh solutions were made before each experiment. One untreated group and one solvent (DMSO and PBS) treated group were always maintained simultaneously.
EXAMPLE 6
In-vivo antileshmanial activity of bioactive fraction and piperine as bioenhancer in the L. donovani infected golden hamster model:
Male 4-6 weeks old Golden hamster (M auratus) were infected with 2.5x107 amastigotes of L. donovani intracardially on day 0 from the spleen of a two months infected donor hamster for infection. The bioactive fraction was dissolved in minimum amount of DMSO and then diluted accordingly with sterile PBS. The bioactive fraction was administered twice per week for three successive weeks intraperitoneally at lOOmg/Kg body weight dose after two weeks of infection. Piperine was administered orally as dry powder two hour before the intraperitoneal injection of bioactive fraction.
Fresh solutions were made before each experiment. One untreated group and one solvent (DMSO and PBS) treated group were always maintained simultaneously.
EXAMPLE 7
In-vivo toxicity testing of hioactive fraction, SAG and bioactive fraction-^ SAG in the
L. donovani infected golden hamster model:
■ Male 4-6 weeks old Golden hamster (M. auratus) were infected with 2.5x107 amastigotes of L. donovani intracardially on day 0 from the spleen of a two months infected donor hamster for infection. The bioactive fraction was dissolved in minimum amount of DMSO and then diluted accordingly with sterile PBS. The bioactive fraction was administered twice per week for three successive weeks intraperitoneally at 200mg/Kg body weight dose, SAG at 5mg/Kg body weight dose and bioactive fraction SAG at 200mg+5mg/Kg body weight dose after eight weeks of infection. Fresh solutions were made before each experiment. One untreated group was always maintained simultaneously. Animals were sacrificed after twelve weeks of infection. Blood was collected and sera isolated for assessment of specific enzyme level as Serum Glutamate Pyruvate Transaminase, Alkaline Phosphatase, Serum Creatinine and Blood Urea. All assay were done using Reddy's Laboratory kit according to manufacturer's protocol.
TABLE 1 : Specific enzyme levels in sera of golden hamster undergoing experimental visceral leishmaniasis indicating non-toxic nature of bioactive fraction
Group and final SGPT Alkaline Phosphatase Serum creatinine Blood Urea
Concentration (Unit/ml) (King-Armstrong unit) (mg %) (mg %)
Normal 49.6il l.63 8.8 ±1.5 0.37±0.06 33.9±2.31
Infected 156.74±10.93 19.98±4.73 0.58±0.11 68.43±13.29
PE treated (200mg/kgBW) 131.37-t27.38 16.3±5.77 0.39±0.04 36.64±5.63
SAG treated (5mg/kgBW) 123.78±27.34 16.9±5.62 0.66±0.07 66.35±13.95
PE+SAG treated 115.33±35.78 19±4.8 0.38±0.06 51.77±9.7
(200mg+5mg/kgBW)
Combined therapy of bioactive fraction and SAG is significantly reducing the SGPT level where p=0.015.
EXAMPLE 8
HPLC (Shimadzu, Japan) chromatogram of bioactive fraction containing calceolarioside A is obtained from methanolic crude extract of night jasmine leaf. The analysis was performed with C18 column (250x4 mm I. D; SGE International PTY, Australia) attached with a Guard column (Supelco) a Rheodyne injection valve with a 100-μl loop and PDA detector (SPD-M 10 Avp). A linear gradient elution was carried out with methanol: water mixtures from 0:100,(v/v) to 100:0,(v/v) within 0 to 60 min at a flow rate of 0.5 ml/min.The column was maintained at room temperature and the detection was performed at UV 210 nm and 254 nm.
EXAMPLE 9
HPLC chromatogram of was performed calceolarioside A from methanolic extract of night jasmine leaf with the same method as mentioned in Example 6.
Advantages:
The main advantages of the present invention are:
1. A potential medicament against kala-azar, visceral leishmaniasis.
2. Herbal antileishmanial chemotherapeutic agent with better activity and less toxicity. 3. Calceolarioside A, novel strikingly potential antileishmanial drug candidate obtained from natural source.
Claims
1. A bioactive fraction obtained from the leaves of the plant night jasmine, wherein the said fraction comprising a) calceolarioside A 39.62% b) kaempferol glycoside 34.8% c) two unidentified compound 5.2% & 5.8% and d) unidentified unresolved fraction 14.6%, wherein said weight percentages are percentages in the total weight of the fraction.
2. A bioactive fraction as claimed in claim 1, wherein the said fraction is useful as a leishmanicidal agent.
3. A bioactive fraction as claimed in claim 1, wherein the said fraction is non toxic to liver and kidney at the dose of 200mg/Kg body weight / day at least for three weeks.
4. Use of the bioactive fraction as claimed in claim 1 in the treatment of visceral leishmaniasis.
5. Use of the compound calceolarioside A as a leishmanicidal agent.
6. Use of the compound calceolarioside A as claimed in claim 5, wherein the said compound reduces the viability of Z. donovani promastigotes in vitro uptolOO % at the dose of 80 μg/ml of culture media for 24 hrs.
7. A pharmaceutical composition useful as a leishmanicidal agent, wherein the said composition comprises a therapeutically effective amount of bioactive fraction as claimed in claim 1 or selected from the combination comprising bioactive fraction and sodium antimony gluconate or bioactive fraction and piperine or bioactive fraction and sodium antimony gluconate and piperine optionally along with one or more pharmaceutically acceptable carriers, additives, lubricant and diluents.
8. A pharmaceutical composition as claimed in claim 7, wherein the additive used are selected from the group comprising of proteins, carbohydrates, sugar, talc, magnesium state, cellulose, calcium, carbohydrate, starch-gelatin paste.
9. A pharmaceutical composition as claimed in claim 7, wherein the said composition further comprising the therapeutically effective amount of bioactive fraction obtained from the leaves of the plant night jasmine optionally along with one or more pharmaceutically acceptable carriers, additives and diluents.
10. A pharmaceutical composition as claimed in claim 9, wherein the dosage of the said composition is administered at a unit dose of at least 100-to 200-mg/Kg- body weight.
11. A pharmaceutical composition as claimed in claim 10, wherein the said composition is preferably administered at a unit dose of 100-mg/Kg body weight at least for three weeks wherein the said composition reduces the parasitic load of spleen up to 81.6%.
12. A pharmaceutical composition as claimed in claim 10, wherein the said composition is preferably administered at a unit dose of 100-mg/Kg body weight at least for three weeks wherein the said composition reduces the parasitic load of liver up to 85.42%.
13. A pharmaceutical composition as claimed in claim 7, wherein the said composition further comprising the therapeutically effective amount of bioactive fraction and sodium antimony gluconate optionally along with one or more pharmaceutically acceptable carriers, additives and diluents wherein the ratio of bioactive fraction and sodium antimony gluconate is in the range of 200:1 to 20:1.
14. A pharmaceutical composition as claimed in claim 13, wherein the dosage of the said composition is administered at a unit dose of at least 150mg/Kg body weight of bioactive fraction and 5 mg/Kg body weight of sodium antimony gluconate to 200mg/Kg body weight of bioactive fraction and 10 mg/Kg body weight of sodium antimony gluconate.
15. A pharmaceutical composition as claimed in claim 14, wherein the dosage of the said composition is preferably administered at a unit dose of 150mg/Kg body weight of bioactive fraction and 5 mg/Kg body weight of sodium antimony gluconate at least for three weeks wherein the said composition reduces the parasitic load of spleen up to 95.1%.
16. A pharmaceutical composition as claimed in claim 14, wherein the dosage of the said composition is preferably administered at a unit dose of 150mg/Kg body weight of bioactive fraction and 5 mg/Kg body weight of sodium antimony gluconate at least for three weeks wherein the said composition reduces the parasitic load of liver up to 99.85%.
17. A pharmaceutical composition as claimed in claim 14, wherein the dosage of the said composition is preferably administered at a unit dose of 200mg/Kg body weight of bioactive fraction and 10 mg/Kg body weight of sodium antimony gluconate at least for three weeks wherein the said composition reduces the parasitic load of liver and spleen up to 100%.
18. A pharmaceutical composition as claimed in claim 7, wherein the said composition further comprising the therapeutically effective amount of bioactive fraction and piperine optionally along with one or more pharmaceutically acceptable carriers, additives and diluents wherein the ratio of bioactive fraction and piperine is in the range of 200: 1 to 20: 1.
19. A pharmaceutical composition as claimed in claim 18, wherein the dosage of the said composition is administered at a unit dose of lOOmg/Kg body weight of bioactive fraction and 0.5 mg/Kg body weight of piperine to 100 mg/Kg body weight of bioactive fraction and 5.0 mg/Kg body weight of piperine.
20. A pharmaceutical composition as claimed in claim 19, wherein the dosage of the said composition is preferably administered at a unit dose, of 100mg/Kg body weight of bioactive fraction and 0.5 mg/Kg body weight of piperine at least for three weeks wherein the said composition reduces the parasitic load of spleen up to 98.8%.
21. A pharmaceutical composition as claimed in claim 19, wherein the dosage of the said composition is preferably administered at a unit dose of 100mg/Kg body weight of bioactive fraction and 0.5 mg/Kg body weight of piperine at least for three weeks wherein the said composition reduces the parasitic load of liver up to 79.8%.
22. A pharmaceutical composition as claimed in claim 19, wherein the dosage of the said composition is preferably administered at a unit dose of 100mg/Kg body weight of bioactive fraction and 5.0 mg/Kg body weight of piperine at least for three weeks wherein the said composition reduces the parasitic load of liver and spleen up to 100%.
23. A pharmaceutical composition as claimed in claim 7, wherein the said composition further comprising the therapeutically effective amount of bioactive fraction and sodium antimony gluconate and piperine optionally along with one or more pharmaceutically acceptable carriers, additives and diluents wherein the ratio of bioactive fraction and sodium antimony gluconate and piperine is in the range of 40: 1: 0.2 to 40: 1: 2 .
24. A pharmaceutical composition as claimed in claim 23, wherein the dosage of the said composition is administered at a unit dose of lOOmg/Kg body weight of bioactive fraction and 2.5 mg/Kg body weight of sodium antimony gluconate and 0.5mg/Kg body weight of piperine to lOOmgKg body weight of bioactive fraction and 2.5 mg/Kg body weight of sodium antimony gluconate and 5.0 mg/Kg body weight of piperine.
25. A pharmaceutical composition as claimed in claim 24, wherein the dosage of the said composition is preferably administered at a unit dose of 100mg/Kg body weight of bioactive fraction and 2.5 mg/Kg body weight of sodium antimony gluconate and 5.0mg/Kg body weight of piperine at least for three weeks wherein the said composition reduces the parasitic load of liver and spleen up to 100%.
26. A method of treating visceral leishmaniasis in a subject, wherein the said method comprising the step of administering to the subject a pharmaceutical composition comprising a therapeutically effective amount of bioactive fraction as claimed in claim 1 or selected from the combination comprising bioactive fraction and sodium antimony gluconate or bioactive fraction and piperine or bioactive fraction and sodium antimony gluconate and piperine optionally along with one or more pharmaceutically acceptable carriers, additives, lubricant and diluents.
27. A method as claimed in claim 26, wherein the said subject is mammal including human.
28. A method as claimed in claim 26, wherein the additive used are selected from the group comprising of proteins, carbohydrates, sugar, talc, magnesium state, cellulose, calcium, carbohydrate, starch-gelatin paste.
29. A method as claimed in claim 26, wherein the said method further comprising the step of administering to the subject a pharmaceutical composition as claimed in claim 9. _
23
30. A method as claimed in claim 29, wherein the said composition is administered at a unit dose of at least 100-to 200-mg/Kg-body weight.
31. A method as claimed in claim 30, wherein the said composition is preferably administered at a unit dose of 100 mg/Kg body weight at least for three weeks wherein the said composition reduces the parasitic load of spleen up to 81.6%.
32. A method as claimed in claim 30, wherein the said composition is preferably administered at a unit dose of 100-mg/Kg body weight at least for three weeks wherein the said composition reduces the parasitic load of liver up to 85.42%.
33. A method as claimed in claim 26, wherein the said method further comprising the step of administering to the subject a pharmaceutical composition as claimed in claim 11.
34. A method as claimed in claim 33, wherein the said composition is administered at a unit dose of at least 150mg/Kg body weight of bioactive fraction and 5 mg/Kg body weight of sodium antimony gluconate to 200mg/Kg body weight of bioactive fraction and 10 mg/Kg body weight of sodium antimony gluconate.
35. A method as claimed in claim 34, wherein the said composition is preferably administered at a unit dose of 150mg/Kg body weight of bioactive fraction and 5 mg/Kg body weight of sodium antimony gluconate at least for three weeks wherein the said composition reduces the parasitic load of spleen up to 95.1%.
36. A method as claimed in claim 34, wherein the said composition is preferably administered at a unit dose of 150mg/Kg body weight of bioactive fraction and 5 mg/Kg body weight of sodium antimony gluconate at least for three weeks wherein the said composition reduces the parasitic load of liver up to 99.85%.
37. A method as claimed in claim 34, wherein the said composition is preferably administered at a unit dose of 200mg/Kg body weight of bioactive fraction and
10 mg/Kg body weight of sodium antimony gluconate at least for three weeks wherein the said composition reduces the parasitic load of liver and spleen up to 100%.
38. A method as claimed in claim 26, wherein the said wherein the said method further comprising the step of administering to the subject a pharmaceutical composition as claimed in claim 13.
39. A method as claimed in claim 38, wherein the said composition is administered at a unit dose of lOOmg/Kg body weight of bioactive fraction and 0.5 mg/Kg body weight of piperine to 100 mg/Kg body weight of bioactive fraction and 5.0 mg/Kg body weight of piperine.
40. A method as claimed in claim 39, wherein the said composition is preferably administered at a unit dose of 100mg/Kg body weight of bioactive fraction and 0.5 mg/Kg body weight of piperine at least for three weeks wherein the said composition reduces the parasitic load of spleen up to 98.8%.
41. A method as claimed in claim 39, wherein the said composition is preferably administered at a unit dose of 100mg/Kg body weight of bioactive fraction and
0.5 mg/Kg body weight of piperine at least for three weeks wherein the said composition reduces the parasitic load of liver up to 79.8%.
42. A method as claimed in claim 39, wherein the said composition is preferably administered at a unit dose of 100mg/Kg body weight of bioactive fraction and 5.0 mg/Kg body weight of piperine at least for three weeks wherein the said composition reduces the parasitic load of liver and spleen up to 100%.
43. A method as claimed in claim 26, wherein the said wherein the said method further comprising the step of administering to the subject a pharmaceutical composition as claimed in claim 21. ,
44. A method as claimed in claim 43, wherein the said composition is administered at a unit dose of 100mg/Kg body weight of bioactive fraction and 2.5 mg/Kg body weight of sodium antimony gluconate and 0.5mg /Kg body weight of piperine tolOOmgKg body weight of bioactive fraction and 2.5 mg/Kg body weight of sodium antimony gluconate and 5.0 mg /Kg body weight of piperine.
45. A method as claimed in claim 44, wherein the said composition is preferably administered at a unit dose of 100mg/Kg body weight of bioactive fraction and 2.5 mg/Kg body weight of sodium antimony gluconate and 5.0mg/Kg body weight of piperine at least for three weeks wherein the said composition reduces the parasitic load of liver and spleen up to 100%.
46. A method as claimed in claim 26, wherein the administration route used is selected from the group consisting of oral, intraperitoneal, intramuscular, intravenous.
47. A composition as claimed in claim7, wherein the composition is in form of capsule, syrup, concentrate, powder and granules.
48. A method for the isolation of compound calceolarioside A from the bioactive fraction of claim 1, wherein the said method comprising the steps of: a. extracting the powdered dry mass of fresh leaves of Nyctanthes arbortristis with alcohol; b. concentrating the extract obtained from step (a) under reduced pressure at atleast 40°C to obtain semi solid mass; c. performing repeatedly chromatography using organic solvent of the said mass as obtained from step (b) to get bioactive fraction; d. performing thin layer chromatography to get desired compound caleorlaroside A.
49. A method as claimed in claim 48, wherein the alcohol used is selected from the group comprising of methanol, ethanol, n-butanol.
50. A method as claimed in claim 48, wherein the semi solid mass of step (b) is chromatographed over a silica gel column having mesh size 60nm-120nm.
51. A method as claimed in claim 48, wherein the organic solvent used is chloroform.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| BRPI0617264-4A BRPI0617264A2 (en) | 2005-10-10 | 2006-10-09 | pharmaceutical composition useful as a leishmanicidal agent |
Applications Claiming Priority (2)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| IN2725DE2005 | 2005-10-10 | ||
| IN2725/DEL/2005 | 2005-10-10 |
Publications (2)
| Publication Number | Publication Date |
|---|---|
| WO2007042902A2 true WO2007042902A2 (en) | 2007-04-19 |
| WO2007042902A3 WO2007042902A3 (en) | 2007-07-05 |
Family
ID=37762083
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| PCT/IB2006/002805 WO2007042902A2 (en) | 2005-10-10 | 2006-10-09 | Extracts from nyctanthes arbortristis for the treatement of leishmaniasis |
Country Status (2)
| Country | Link |
|---|---|
| BR (1) | BRPI0617264A2 (en) |
| WO (1) | WO2007042902A2 (en) |
Cited By (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN101816665A (en) * | 2010-03-18 | 2010-09-01 | 长春理工大学 | Immunosuppressive medicament |
| EP2805722A4 (en) * | 2012-01-19 | 2015-09-02 | Suntory Holdings Ltd | OLIVE EXTRACT CONTAINING DE (RHAMNOSYL) -ACTOSIDE |
| US20160158302A1 (en) * | 2014-12-06 | 2016-06-09 | B. Arvind Shah | Regulation of Brain Biogenic Amines Associated with Depression by a Formulation from Botanical Source |
-
2006
- 2006-10-09 BR BRPI0617264-4A patent/BRPI0617264A2/en not_active Application Discontinuation
- 2006-10-09 WO PCT/IB2006/002805 patent/WO2007042902A2/en active Application Filing
Non-Patent Citations (10)
| Title |
|---|
| CHAN-BACAB M J ET AL: "Plant natural products with leishmanicidal activity." NATURAL PRODUCT REPORTS DEC 2001, vol. 18, no. 6, December 2001 (2001-12), pages 674-688, XP002422232 ISSN: 0265-0568 * |
| KAPIL A: "Piperine: a potent inhibitor of Leishmania donovani promastigotes in vitro." PLANTA MEDICA, vol. 59, no. 5, October 1993 (1993-10), page 474, XP002423245 ISSN: 0032-0943 * |
| MATHURAM, V. ET AL: "Occurrence of desrhamnosylverbascoside in Nyctanthes arbor-tristis and NMR studies of its acetate" JOURNAL OF THE INDIAN CHEMICAL SOCIETY, vol. 71, no. 4, 1994, pages 215-217, XP009079731 ISSN: 0019-4522 * |
| NAIR R ET AL: "Antibacterial activity of some selected Indian medicinal flora" TURKISH JOURNAL OF BIOLOGY, vol. 29, no. 1, February 2005 (2005-02), pages 41-47, XP002422231 ISSN: 1300-0152 * |
| PURI A ET AL: "Immunostimulant activity of Nyctanthes arbor-tristis L." JOURNAL OF ETHNOPHARMACOLOGY, vol. 42, no. 1, March 1994 (1994-03), pages 31-37, XP002422364 ISSN: 0378-8741 * |
| RATHORE A ET AL: "AN IRIDOID GLUCOSIDE FROM NYCTANTHES-ARBORTRISTIS" PHYTOCHEMISTRY, vol. 28, no. 7, 1989, pages 1913-1918, XP002422228 ISSN: 0031-9422 * |
| SINGHA U K ET AL: "ANTILEISHMANIAL ACTIVITY OF TRADITIONAL PLANTS AGAINST LEISHMANIA DONOVANI IN GOLDEN HAMSTERS" INTERNATIONAL JOURNAL OF PHARMACOGNOSY, vol. 30, no. 4, 1992, pages 289-295, XP009079728 ISSN: 0925-1618 * |
| TANDON J S ET AL: "Iridoids: a new class of leishmanicidal agents from Nyctanthes arbortristis." JOURNAL OF NATURAL PRODUCTS, vol. 54, no. 4, July 1991 (1991-07), pages 1102-1104, XP002915617 ISSN: 0163-3864 cited in the application * |
| VEERAREDDY, P. R. ET AL: "Formulation and evaluation of oil-in-water emulsions of piperine in visceral leishmaniasis" PHARMAZIE, vol. 59, no. 3, March 2004 (2004-03), pages 194-197, XP002423246 ISSN: 0031-7144 * |
| WORTMANN, G ET AL: "A randomized, double-blind study of the efficacy of a 10- or 20-day course of sodium stibogluconate for treatment of cutaneous leishmaniasis in United States military personnel" CLINICAL INFECTIOUS DISEASES, vol. 35, no. 3, 1 August 2002 (2002-08-01), pages 261-267, XP002423244 ISSN: 1058-4838 * |
Cited By (5)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN101816665A (en) * | 2010-03-18 | 2010-09-01 | 长春理工大学 | Immunosuppressive medicament |
| EP2805722A4 (en) * | 2012-01-19 | 2015-09-02 | Suntory Holdings Ltd | OLIVE EXTRACT CONTAINING DE (RHAMNOSYL) -ACTOSIDE |
| AU2013210416B2 (en) * | 2012-01-19 | 2017-03-23 | Suntory Holdings Limited | Desrhamnosyl acteoside-containing olive extract |
| US20160158302A1 (en) * | 2014-12-06 | 2016-06-09 | B. Arvind Shah | Regulation of Brain Biogenic Amines Associated with Depression by a Formulation from Botanical Source |
| US10111921B2 (en) | 2014-12-06 | 2018-10-30 | Govind Prasad Dubey | Regulation of brain biogenic amines associated with depression by a formulation from botanical source |
Also Published As
| Publication number | Publication date |
|---|---|
| BRPI0617264A2 (en) | 2011-07-19 |
| WO2007042902A3 (en) | 2007-07-05 |
Similar Documents
| Publication | Publication Date | Title |
|---|---|---|
| US6531505B2 (en) | Immunosuppressive agents | |
| DE69837551T2 (en) | Composition and method for treating Alzheimer's disease and other amyloidoses | |
| JP4909984B2 (en) | New uses for lignan compounds | |
| US20070292539A1 (en) | Anti-inflammatory cranberry flavonol extract preparations | |
| US20100063153A1 (en) | Anti-cholesterolemic compounds and methods of use | |
| NZ544691A (en) | Methods of isolating amyloid-inhibiting compounds and use of compounds isolated from Uncaria tomentosa and related plants | |
| JPH06502413A (en) | Proanthocyanidin polymer with antiviral activity and method for producing the same | |
| US8293292B2 (en) | Extract of Fraxinus excelsior seeds and therapeutic applications therefor | |
| KR100228510B1 (en) | A process for the preparation of ginsenoside Rg3 and/or Rg5 | |
| JPH0543469A (en) | Beta-glucuronidase inhibitor | |
| WO2010032269A2 (en) | Anti-inflammatory activity of the iridoid glycosides | |
| WO2005003145A1 (en) | Shanzhuyu extract and uses thereof | |
| JPH01157995A (en) | Internal ester of genglioside having analgesic-anti-inflammatory activity | |
| NO343552B1 (en) | Compositions for the treatment of chronic degenerative inflammatory conditions | |
| US20120308677A1 (en) | Pharmaceutical Composition for Preventing and Treating Inflammatory Diseases Containing an Ethyl Acetate Fraction of Dried Extract of Trachelospermi Caulis as an Active Ingredient, and Method for Producing the Fraction | |
| EP3156058A1 (en) | Anti-tumor pharmaceutical application of pentacyclic triterpene saponin compounds of szechuan melandium root | |
| CN113773409B (en) | Polysaccharides from Scutellaria saponifera and its application | |
| WO2007042902A2 (en) | Extracts from nyctanthes arbortristis for the treatement of leishmaniasis | |
| CN102526157B (en) | Application of safflower extract to prevention or treatment of neurodegeneration disease | |
| CN101588810A (en) | Composition comprising trachelospermi caulis and pyrola japonica extracts for treating and preventing inflammatory diseases | |
| KR100337471B1 (en) | Kidney Protection Composition in Processed Ginseng Extract | |
| KR100207293B1 (en) | Immunostimulator Compositions Containing Mixed Water Extracts of Hwangbaekpi and Matari Plants | |
| CN109364063B (en) | Compound with antibacterial effect and application thereof in preparation of antibacterial drugs | |
| JPH06172195A (en) | UDP-glucuronyl transferase inhibitor | |
| JPH0717857A (en) | Cardiovascular drug |
Legal Events
| Date | Code | Title | Description |
|---|---|---|---|
| 121 | Ep: the epo has been informed by wipo that ep was designated in this application | ||
| DPE1 | Request for preliminary examination filed after expiration of 19th month from priority date (pct application filed from 20040101) | ||
| NENP | Non-entry into the national phase |
Ref country code: DE |
|
| 122 | Ep: pct application non-entry in european phase |
Ref document number: 06820782 Country of ref document: EP Kind code of ref document: A2 |
|
| ENP | Entry into the national phase |
Ref document number: PI0617264 Country of ref document: BR Kind code of ref document: A2 Effective date: 20080410 |