WO2005077392A1 - Formulation a base de plantes medicinales comprenant des extraits de withania, tinospora and picrorhiza servant de tonique en pediatrie - Google Patents

Formulation a base de plantes medicinales comprenant des extraits de withania, tinospora and picrorhiza servant de tonique en pediatrie Download PDF

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WO2005077392A1
WO2005077392A1 PCT/IB2005/000133 IB2005000133W WO2005077392A1 WO 2005077392 A1 WO2005077392 A1 WO 2005077392A1 IB 2005000133 W IB2005000133 W IB 2005000133W WO 2005077392 A1 WO2005077392 A1 WO 2005077392A1
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extract
extracts
withania
picrorhiza
tinospora
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PCT/IB2005/000133
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English (en)
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Rahul Singh
Aravind Padiyar
Anil Kanaujia
Yogita Rani
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Ranbaxy Laboratories Limited
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    • 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)
    • A61K36/59—Menispermaceae (Moonseed family), e.g. hyperbaena or coralbead
    • 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)
    • A61K36/80—Scrophulariaceae (Figwort family)
    • 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)
    • A61K36/81—Solanaceae (Potato family), e.g. tobacco, nightshade, tomato, belladonna, capsicum or jimsonweed

Definitions

  • the present invention relates to a new herbal pharmaceutical formulation useful as a pediatric tonic, processes for its preparation, and quantitative analysis of the individual phytochemicals contained in the formulation.
  • the herbal formulation is made up of three plants namely, Withania somnifera (Linn.) Dunal, Tinospora cordifolia (Willd.) Miers and Picrorhiza kurroa Royle.
  • Background of the Invention Ensuring healthy child development is a fundamental step in building the health of a nation. The two most important aspects that can directly or indirectly have adverse effects on the growth of a child are a lack of resistance to fight against diseases and an insufficient intake of food due to decreased appetite.
  • humans are bombarded with pathogens, most of which the body can handle.
  • the commonly used herbs which have been found to possess these properties are: Satavari (Asparagus racemosus); Bala (Sida cordifolia); Draksha (Vitis viniferd); Brahmi (Bacopa monnieri); Ashwagandha (Withania somnifera); Jivanti (Leptadenia reticulata); Amalaki (Emblica officinalis); Guduchi (Tinospora cordifolia); Shatpushpa (Anethum sowa); Atibala (Abutilon indicum); Dadima (Punica granatum); Nagarmotha (Cyperus rotundus); Ativisha (Aconiturn ferox); Daruharidra (Berberis aristatd); Bhringaraja (Eclipta alba); Aj oda (Carum roxburghianum); Sunthi (Zingiber ojficinale); Sariva (Hemidesmus indicus); Vidanga (Embelia
  • Withania somnifera (Linn.) Dunal: (Synonym: Physalis somnifera Linn.), Family: Solanaceae This plant is widely distributed in North- Western and Central India - Mumbai, Bengal, Bengal, Madhya Pradesh, Tamil Pradesh and Bengal in waste places and on bunds. It now also is widely cultivated in certain areas of Madhya Pradesh and Bengal. The chemistry of Withania somnifera has been extensively studied and over 35 chemical constituents have been identified, extracted and isolated.
  • the biologically active chemical constituents of roots are alkaloids - tropine, pseudotropine, 3- ⁇ - tigloyloxytropane, cuscohygrine, dl-isopelletierine, anahygrine, hygrine, withasomnine (Schroter et al, Tetrahedron 22, 2895-2897, 1966), steroidal lactones - withanolides, withaferins and glycowithanolides - sitoindoside IX and X (Ghosal et al., Planta Medica. 54, 561, 1988). Withania somnifera is also rich in iron.
  • Tinospora cordifolia (willd.)
  • Miers (Synonym :Menispermum cordifolium Willd.), Family: Menispermaceae This Plant runs over the tops of high trees and shrubs. It is wild in forest areas of Bundelkhand, Central India, Maharashtra, Tamil (Dang), Konkan and other tropical and sub-tropical parts of India. Sometimes it is cultivated as an ornamental plant in gardens and for medicinal value. Phytochemical investigations of T.
  • cordifolia show the presence of diterpenoid furanolactones and their glycosides viz., tinosporide, palmatosides, cordioside, tinosponone, tinocordioside, menispermacide, columbin and tinosporaside (Maurya et al., Phytochemistry 38, 659-661, 1995); norditerpene furan glycosides viz., cordifoliosides A- E (Gangan et al., Phytochemistry 39, 1139-1142, 1995); sesquiterpenoid viz., tinocordifolin and its glycoside, tinocorifolioside (Maurya et al., Phytochemistry 49, 1343- 1345, 1998); and steroids viz., ⁇ -sitosterol, ecdysones and maikisterone A (Gangan et al., Indian J.
  • T. cordifolia is very well known, is a highly-praised herb of the Indian system of medicine and enjoys the reputation of being a Rasayana (Adaptogen) drug. In fact, its use as a Rasayana is mentioned in Charaka Samhita (1,000 BC). It also is believed to be an excellent immuno-modulator. The experimental work also strengthens the age-old claims. It has been found to be an excellent immuno-modulator, anti-oxidant and hepato- protective agent. Studies found that T. cordifolia aqueous extract in a dose of 400 mg/kg/day in sub acute toxicity studies was safe (Grover et al., J. Ethnopharmacology 73; 461-70, 2000).
  • Picrorhiza kurroa Royle, Family Scrophulariaceae Picrorhiza is found in the North-Western Himalayas from Kashmir to Kumaon up to an altitude of approximately 4000 meters in India.
  • the main active constituent of the roots of P. kurroa is Kutkin - a bitter glycosidal principle.
  • Kutkin was later shown to be a stable mixed crystal of two C-9 iridoid glycosides — Picroside I [6'-O-trans-cinnamoyl-catalpol] and Kutakoside [10-O- vanilloylcatalpol] (Jia et al., J. Natural Products 62, 901- 903, 1999).
  • Picroside II (6-O-vanilloylcatalpol) and Picroside III (6'-[4-hydroxy-3- methoxycinnamoyl] catalpol were also isolated from the roots. Apocynin is also reported to be isolated from these plant roots (Laurie et al., Phytochemistry 24, 2659-2661, 1985 and Rastogi et al., Compendium of Indian Medicinal Plants. Publication and Information Directorate, CSIR, New Delhi. 2, 536, 1993). P. kurroa has traditionally been used to treat hepatic disorders. Hence, many experimental studies were aimed to evaluate its hepato-protective as well as hepato- curative effect.
  • mice with 0.1, 1.0 and 10.0 mg/kg body weight per os did not enhance the magnitude of chromosomal aberrations and therefore Picroliv seems to be devoid of clastogenic activity (Jain & Sethi, Fitopad., 63; 255-257, 1992).
  • Ayurvedic preparations available in markets all over the world for use as pediatric tonics Lower side effects, varied medicinal values and ability to completely cure a particular ailment are the positive aspects of Ayurvedic medicines in comparison to Allopathic medicines, which tend to have pronounced side effects, limited medicinal value and symptomatic treatment of ailment.
  • a herbal pharmaceutical formulation that includes extracts of Withania, Tinospora and Picrorhiza, or a mixture of active ingredients from these herbs, and one or more pharmaceutically acceptable carriers and/or diluents.
  • Embodiments of the herbal pharmaceutical formulation may include one or more of the following features.
  • the percentage w/v of Picroside I may be from about 6.62 x 10-3 to about 6.34 x 10-3.
  • the percentage w/v of Cordifolioside A may be from about 2.77 x 10-3 to about 2.56 x 10-3.
  • the percentage w/v of Withaferin A may be from about 2.89 x 10-3 to about 1.96 x 10-3.
  • the extracts of Withania, Tinospora and Picrorhiza may make up 1.0 - 5.0% w/v, 0.1 - 0.6% w/v, and 0.1 - 0.7% w/v of the formulation, respectively.
  • the formulation may include the extracts of Withania somnifera (Linn.) Dunal,
  • Tinospora cordifolia (Willd.) Miers and Picrorhiza Kurroa Royle.
  • a method of making a pharmaceutical formulation that includes extracts of Withania, Tinospora and Picrorhiza, or a mixture of active ingredients from these herbs, and one or more pharmaceutically acceptable carriers and/or diluents.
  • the process includes (a) mixing the individual extracts of Withania,
  • Tinospora, and Picrorhiza in therapeutically effective amounts to get a combined extract; (b) adding one or more pharmaceutically acceptable carriers and/or diluents; and (c) optionally, filtering the final mixture.
  • a process for the isolation of Withaferin A, Cordifolioside A and Picroside I from the plants Withania somnifera, Tinospora cordifolia and Picrorhiza kurroa respectively.
  • the process includes (a) extracting a powdered part of the respective plant with a suitable organic solvent optionally containing water; (b) concentrating the extracts obtained in step a); (c) extracting the concentrated extracts of step b) with second organic solvents and, optionally, filtering the solution to remove any non-polar impurities; (d) purifying the resultant organic extracts of step c) by chromatographic technique; and (e) isolating the phytochemical marker compounds by optional crystallization.
  • Embodiments of the process may include one or more of the following features.
  • the suitable organic solvent may be one or more lower alkanols and the second organic solvent may be one or more non-polar aliphatics or cycloalkyls, ethers, esters, chlorinated hydrocarbons, ketones, or mixtures thereof.
  • the chromatographic technique may be one or more of column chromatography, TLC, HPTLC and HPLC.
  • the process purification of the organic extract may include the use of a silica gel 230-400 mesh or alumina in the chromatographic technique.
  • the process may further include, or as a separately process include, identifying the Withaferin A, Cordifolioside A or Picroside I in their respective plant extracts.
  • the process of identifying includes a.
  • the plant extracts may be selected from the extracts of
  • the reference standard solution may be prepared by dissolving Withaferin A, Cordifolioside A and Picroside I separately in suitable organic solvents and, optionally, filtering the solution.
  • the test solution may be prepared by dissolving the extract of Withania, Tinospora and Picrorhiza separately in suitable organic solvent and, optionally, filtering the solution.
  • the process may further include, or as a separately process include, estimating a total Withanolide content in Withania extract.
  • the estimating process includes: a. diluting the extract with polar solvent optionally containing water to form a mixture, sonicating the mixture, and filtering the mixture; b.
  • the Withania extract may contain Withanolide content from about 0.05% to about 25%.
  • the process may further include, or as a separately process include, estimating a total bitters content in Tinospora or Picrorhiza extract.
  • the estimating includes: a. diluting the extract with polar solvent optionally containing water, refluxing, decanting to remove the supernatant, and filtering to obtain a residue; b.
  • the Tinospora extract may include a total bitter content of about 0.1 % to about 50 % and the Picrorhiza extract may include a total bitter content of about 0.05 % to about 25 %.
  • a method for the detection and quantification of Withaferin A, Cordifolioside A and Picroside I in a herbal formulation that includes extracts of Withania, Tinospora and Picrorhiza, or a mixture of active ingredients that have been extracted from these herbs, and pharmaceutically acceptable carriers and or diluents.
  • the method of detection and quantification includes: a. extracting the formulation with one or more organic solvents which optionally contains water; b. concentrating the extract of step a) and reconstituting the residue in one or more second organic solvents; c. spotting the solution obtained in step b) against each standard phytochemical marker on a HPTLC plate; d.
  • each of the standard phytochemical marker may be prepared by dissolving Withaferin A, Cordifolioside A and Picroside I separately in second organic solvent and optionally filtering the solution.
  • the wavelength in step f) used may be from about 286 nm to about 636 nm.
  • the wavelength used in step f) maybe 286 nm, 606 nm or 636 nm for Picroside I, Withaferin A or Cordifolioside A, respectively.
  • the details of one or more embodiments of the inventions are set forth in the description below. Other features, objects and advantages of the inventions will be apparent from the description and claims.
  • the formulation is believed to be an excellent pediatric tonic, which ensures optimum growth, immunity and enhanced appetite and at the same time is devoid of toxic side effects.
  • Figure 1 shows TLC photograph of Withaferin A from the extracts of Withania against Standard Withaferin A.
  • Figure 2 shows TLC photograph of Cordifolioside A from the extracts of Tinospora against Standard Cordifolioside A.
  • FIG. 3 shows TLC photograph of Picroside I from the extracts of Picrorhiza against Standard Picroside I.
  • FIG 4 shows TLC photograph of Withaferin A, Cordifolioside A and Picroside I extracted from the syrup against Standard phytochemical markers.
  • Figure 5 shows HPTLC chromatogram of Withaferin A extracted from the syrup against standard Withaferin A.
  • Figure 6 shows HPTLC chromatogram of Cordifolioside A extracted from the syrup against standard Cordifolioside A.
  • Figure 7 shows HPTLC chromatogram of Picroside I extracted from the syrup against standard Picroside I.
  • the present inventors have developed a reasoned belief that Withania, Tinospora and Picrorhiza, which are traditionally known to have excellent medicinal values, after combining will provide an advanced formulation useful as a tonic.
  • the formulation which is devoid of side effects, can be used in pediatric patients for overall growth, immunity and for increasing their appetite.
  • the present inventors have also felt a need to standardize the formulation by adopting certain strict criterion for selecting raw materials, processing the extraction of the herbs, and manufacturing the formulation.
  • the present inventors have embarked upon a quantification method of the active phytochemicals present in the formulation, which will help provide for a rational use of the formulation.
  • the present invention provides new herbal pharmaceutical formulations which include extracts of Withania, Tinospora and Picrorhiza or a mixture of active ingredients from these herbs, methods of preparing the pharmaceutical formulations, and methods of isolation, identification, quantification, and/or estimation of individual phytochemicals from their respective plants separately and in the new pharmaceutical formulations.
  • the first aspect of the invention provides a new herbal pharmaceutical formulation that is made up of extracts of Withania, Tinospora and Picrorhiza, or a mixture of active ingredients that have been extracted from these herbs, with pharmaceutically acceptable carriers and/or diluents.
  • the second aspect of the invention provides a process for the isolation of the phytochemical marker compounds viz., Withaferin A of Formula I
  • FORMULA III each from the plants Withania somnifera, Tinospora cordifolia and Picrorhiza kurroa, respectively.
  • the process includes a) extracting the powdered selected part of the plant with a suitable organic solvent optionally containing water; b) concentrating the extracts obtained in step a); c) extracting the concentrated extracts of step b) with one or more second organic solvents and, if required, filtering the solution to remove the non-polar impurities; d) purifying the resultant organic extracts of step c) by chromatographic technique; and e) isolating the phytochemical marker compounds by optional crystallization;
  • the phytochemical markers or marker compounds are present in higher concentration in particular parts of the plant depending on the plant. For example, in the case of
  • Withania somnifera the phytochemical marker - Withanolide A
  • the powdered roots of Withania somnifera wherein the particle size of the powder is NMT 30 # is extracted three times with a suitable organic solvent selected from water, soluble lower alkanols such as methanol, ethanol, n-propanol and isopropanol, or mixtures thereof at a temperature between 35°C to 80°C. It is preferred to extract the powder at reflux temperature of the solvent used. The combined extracts are then concentrated under reduced pressure.
  • the concentrated extracts next are mixed with water and the aqueous layer is washed with one or more second organic solvents selected from non-polar aliphatic or cycloalkyl solvents such as hexane, heptane, petroleum ether, cyclohexane; ethers such as diethyl ether, diisopropyl ether; esters such as ethyl acetate, methyl acetate, ethyl formate, methyl formate, isobutyl acetate, n-butyl acetate; chlorinated hydrocarbons such as methylene chloride, ethylene chloride, chloroform and carbon tetrachloride; ketones such as acetone, ethyl methyl ketone, diisobutyl ketone, methyl isobutyl ketone; or mixtures thereof.
  • non-polar aliphatic or cycloalkyl solvents such as hexane, heptane, petroleum
  • the organic washings are pooled together, dried over a suitable drying agent and filtered.
  • the organic extracts are then further concentrated under reduced pressure.
  • the concentrated extract is then subjected to column chromatographic purification through an alumina column and first eluted with petroleum ether and then with an increasing quantity of ethyl acetate in petroleum ether.
  • the fractions are collected separately and scanned for Withaferin A presence by TLC using chloroform:methanol : : 9: 1 mobile phase and anisaldehyde/sulphuric acid as the developing spray.
  • the mobile phase can also be selected from one or more second organic solvents.
  • Sulphuric acid and glacial acetic acid can also be used alone or in combination with anisaldehyde as a detecting/developing spray or reagent.
  • the fractions having Withaferin A as observed by TLC pattern are combined and concentrated under vacuum.
  • the crude Withaferin A is then crystallized from ethyl acetate and has melting point of about 241-243°C.
  • the phytochemical marker - Cordifolioside A is present in the stems or bark of the plant.
  • the powdered fresh stems wherein the particle size of the powder is NMT 30 # is extracted in a similar manner as described above in the case of Withania.
  • Cordifolioside A is treated with acetic anhydride to get Cordifolioside A tetra-acetate which is purified from methanol and has a melting point of about 140°C.
  • the phytochemical marker - Picroside I is present in the roots of the plant.
  • the powdered fresh roots wherein the particle size of the powder is NMT 30 # is extracted in a similar manner as described above in the case of Withania. After concentration of the extracts, water is added and the aqueous layer is washed with hexane and then extracted three times with equal volumes of ethyl acetate. The combined extracts are dried over a suitable drying agent and then concentrated under reduced pressure.
  • the third aspect of the invention provides a method of identification of Withaferin A, Cordifolioside A, or Picroside I in their respective plant extracts.
  • the method includes the steps of: a. applying the reference standard and test solution to the chromatoplate; b.
  • step (c) developing the chromatoplate of step (a) in a suitable mobile phase; c. drying the chromatoplate of step (b) and if required, spraying it with asuitable developing reagent; and d. identifying the Withaferin A, Cordifolioside A or Picroside I after step (c).
  • Plant extracts can be selected from the extract of Withania somnifera, Tinospora cordifolia or Picrorhiza kurroa.
  • the reference standard solutions can be prepared by dissolving Withaferin A, Cordifolioside A or Picroside I, separately, in suitable organic solvent and, optionally, filtering the solution.
  • the test solution can be prepared by dissolving the extract of Withania, Tinospora or Picrorhiza separately in a suitable organic solvent and optionally filtering the solution.
  • the suitable organic solvent of this aspect includes lower alkanols such as methanol, ethanol, n-propanol, isopropanol or mixtures thereof.
  • methanol is used for the purpose.
  • the suitable mobile phase of this aspect includes alcohols such as methanol, ethanol, n-propanol, isopropanol; non-polar aliphatic or cycloalkyl solvents such as hexane, heptane, petroleum ether, cyclohexane; ethers such as diethyl ether, diisopropyl ether; esters such as ethyl acetate, methyl acetate, ethyl formate, methyl formate, isobutyl acetate, n-butyl acetate; chlorinated hydrocarbons such as methylene chloride, ethylene chloride, chloroform and carbon tetrachloride; ketones such as acetone, ethyl methyl ketone, diisobutyl ketone, methyl isobutyl ketone; and mixtures thereof.
  • alcohols such as methanol, ethanol, n-propanol, isopropanol
  • the photographs in Figure 1, Figure 2 and Figure 3 as shown in the accompanied drawing show spots corresponding to Withaferin A, Cordifolioside A and Picroside I, respectively, from the respective extracts having the same Rf-values when compared with the standard marker compounds.
  • the fourth aspect of the present invention provides a method for estimating the total Withanolide content in Withania extract.
  • the method includes a. diluting the Withania extract with polar solvent optionally containing water, sonicating the mixture obtained, and filtering it; b. extracting the filtrate of step (a) with non-polar solvent to remove the non-polar components from the polar solvent extract; c. extracting the polar solvent extract obtained in step (b) with ether; and d.
  • the polar solvent of this aspect can be selected from one or more alcohols including methanol, ethanol, n-propanol, isopropanol or mixtures thereof. Methanol is one preferred polar solvent.
  • the non-polar solvent of this aspect can be selected from one or more aliphatic or cycloalkyl solvents including hexane, heptane, petroleum ether, cyclohexane or mixtures thereof. Hexane is one preferred non-polar solvent.
  • the ether solvent of this aspect can be one or more ethers including diethyl ether, diisopropyl ether or mixtures thereof.
  • Diethyl ether is one preferred ether.
  • the Withania extract is diluted with methanol and water and the resulting mass is sonicated and filtered.
  • the clear filtrate is extracted with hexane for a minimum of four times and the resulting hexane extract is discarded.
  • the aqueous methanolic layer is then extracted with diethyl ether for a minimum of four times and the diethyl ether layer is washed with water. After drying the diethyl ether layer over sodium sulphate, it is concentrated under a reduced pressure in a pre- weighed round bottom flask. After complete concentration, the round bottom flask is dried and cooled in a desiccator and weighed.
  • the fifth aspect of the invention provides a method for estimating the total bitters content in Tinospora extract.
  • the method includes a. diluting the Tinospora extract with polar solvent optionally containing water, refluxing it, decanting it to remove the supernatant, and filtering it; b. extracting the residue of step (a) with polar solvent and combining the resultant extracts; c. concentrating the resultant extract of step (b), dissolving it in hot water and repeatedly washing the hot water extract with an organic solvent; and d. separating/collecting the organic solvent extract of step (c), concentrating the extract, and drying the extract to calculate the percentage content of total bitters in the extract.
  • the polar solvent of this aspect can be selected from one or more alcohols including methanol, ethanol, ⁇ -propanol, isopropanol or mixtures thereof. Methanol is one preferred polar solvent.
  • the organic solvent of this aspect can be selected from one or more esters including ethyl acetate, methyl acetate, ethyl formate, methyl formate, isobutyl acetate, n- butyl acetate or mixtures thereof. Ethyl acetate alone is one preferred organic solvent.
  • the extract is taken in methanol and refluxed on a water bath. The mass after cooling is decanted to remove the supernatant and filtered through filter paper. The residue is further extracted with methanol until the extract becomes colorless.
  • the sixth aspect of the present invention provides a method for estimating the total bitters content in Picrorhiza extracts.
  • the Picrorhiza extract is processed exactly as per the process mentioned in the fifth aspect of the invention and then the total bitters are calculated by using Formula II as mentioned above.
  • the seventh aspect of the present invention provides a process for preparating a herbal pharmaceutical formulation that includes extracts of Withania, Tinospora and Picrorhiza or a mixture of active ingredients from these herbs.
  • the pharmaceutical formulation further includes one or more pharmaceutically acceptable carriers and/or diluents.
  • the process includes a. mixing the individual extract of Withania, Tinospora and Picrorhiza in therapeutically effective amounts to get a combined extract; b.
  • a pharmaceutically acceptable carrier such as water miscible pharmaceutically acceptable co-solvents including propylene glycol, sorbitol, glycerol, polyethylene glycol and the like or mixture thereof.
  • Purified water can also be added to the extract.
  • the resultant mixture is then optionally filtered through a Sparkler Filter to remove insoluble foreign particulate contamination.
  • a pharmaceutically acceptable preservative such as propyl paraben, methyl paraben, and quaternary ammonium salts such as cetrimide and the like or a mixture thereof along with suitable flavoring agent and a sequestering agent.
  • the formulation can be made palatable by adding to it a sweetening agent such as invert sugar syrup, xylitol, mannitol, sugar syrup, jaggery, honey, maltose syrup, or mixtures thereof.
  • Non-sugar-based sweetening agents also can be used.
  • the eighth aspect of the present invention provides a method for the detection and quantification of Withaferin A, Cordifolioside A and Picroside I in a herbal formulation that includes extracts of Withania, Tinospora and Picrorhiza or a mixture of active ingredients that have been extracted from these herbs and also includes one or more pharmaceutically acceptable carriers and/or diluents.
  • the method includes a. extracting the formulation with an organic solvent or a mixture of organic solvents which optionally contains water; b.
  • step (a) concentrating the extract of step (a) and reconstituting the residue in a second organic solvent; c. spotting the solution obtained in step (b) against each standard phytochemical marker on a HPTLC plate; d. developing the HPTLC plate of step (c) in a mobile phase; e. spraying or applying the detection/developing reagent on the HPTLC plate of step (d); f. scanning the plate of step (e) at particular wavelengths; and g. detecting and quantifying Withaferin A, Cordifolioside A and Picroside I after step (f).
  • the oral syrup prepared by the process of the seventh aspect of the invention is further diluted with distilled water and extracted with an organic solvent or mixture of organic solvents.
  • the organic extracts obtained are dried over a suitable drying agent, then concentrated under vacuum to remove the organic solvent, and the residue obtained is reconstituted in a second organic solvent or mixture of solvents.
  • the test solution thus obtained is spotted against the standard phytochemical marker solutions on HPTLC plates and the plates are allowed to develop in a saturated chamber that has a mobile phase. After complete development, the plates are sprayed with a solution of anisaldehyde in sulphuric acid. Sulphuric acid, glacial acetic acid, anisaldehyde or mixtures thereof can also be used as developing/detecting reagents.
  • the plates are then dried and scanned at various wavelengths to detect and quantify the phytochemical markers present in the formulation against standard phytochemical markers.
  • the organic solvent is selected from the groups comprising alkanols such as methanol, ethanol, n-propanol, isopropanol, n-butanol, isobutanol, tert-butanol and n- octanol; ketones such as acetone, ethyl methyl ketone, methyl isobutyl ketone and diisobutyl ketone; esters such as methyl formate, ethyl formate, methyl acetate, ethyl acetate, n-propyl acetate, isopropyl acetate, n-butyl acetate, isobutyl acetate and tert-butyl acetate; chlorinated hydrocarbons such as methylene chloride, chloroform, carbon tetrachloride and ethylene chloride; aromatic hydrocarbons such as xylene, toluene, benzene, substituted benzen
  • the second organic solvent may include some of those solvents described above, including methanol, ethanol, isopropanol, n-propanol, and acetone. Besides these acetonitrile, diethyl ether, N,N-dimethylformamide, N,N-dimethylacetamide, dimethylsulphoxide and tetrahydrofuran may also be used.
  • the standard phytochemical marker solutions are prepared by dissolving a fixed quantity of each of Withaferin A, Cordifolioside A or Picroside I independently in a fixed quantity of the second organic solvent mentioned above. The solution can be sonicated and then made up to a desired fixed volume using the same solvent. These standard solutions are then used as the Reference Standard Solutions.
  • HPTLC is performed using silica gel 60 F 54 pre-coated Aluminium foil plate with a layer thickness of 0.2 mm (1.05554, Merck). Reference and test solutions are applied on the plates in a fixed volume using a hypodermic syringe. The distance between two spots is kept about 10 mm having a band length of about 8 mm.
  • the mobile phase used can be selected from a mixture of solvents described as the first organic solvent. A preferred mobile phase includes chloroform and methanol in a preferred ratio of 85:15.
  • the mobile phase is placed in a glass chamber. After saturating the glass chamber with the mobile phase, the HPTLC plates are placed into the chamber and the development distance of 8 cm from point of application is selected.
  • the plate is removed from the chamber and dried using a hot air blower. After spraying the plate with anisaldehyde in a sulphuric acid reagent, the plate is dried and scanned at three wavelengths: (1) 286 nm for determination of Picroside I, (2) 606 nm for determination of Withaferin A, and (3) 636 nm for detection of
  • ASPL Area of test sample corresponding to phytochemical marker
  • the ninth aspect of the present invention provides a method of ensuring optimum growth and immunity as well as a method increasing the appetite.
  • the method includes administering a herbal formulation orally to a child in need thereof, the herbal formulation including extracts of Withania, Tinospora and Picrorhiza, or a mixture of active ingredients that have been extracted from these herbs, and pharmaceutically acceptable carriers and/or diluents. While the following examples are provided to certain embodiments of the invention, they are not intended to be limiting to the scope of the invention. EXAMPLE 1
  • the material thus obtained was chromatographed through an alumina column and eluted first with petroleum ether and then with an increasing volume of ethyl acetate in petroleum ether and the different fractions were collected. Each fraction was observed by TLC using mobile phase CHC1 3 : MeOH :: 90 : 10 and detected by spraying with anisaldehyde/sulphuric acid reagent. All the fractions having Withaferin A as observed by their TLC pattern were combined. Then, they were concentrated under reduced pressure and Withaferin A was crystallized from ethyl acetate.
  • Withaferin A Two milligrams of Withaferin A reference standard was accurately weighed and transferred to a 5 ml volumetric flask. Three milliliters methanol was added and sonicated in an ultrasonic water bath to dissolve. The volume was made up with methanol. The resulting solution was used as a reference standard solution for Withaferin A.
  • this material may be filtered through cotton/filter paper and the resulting aqueous extract transferred into a 250 ml separating funnel. The residue was washed with 10-15 ml hot water and transferred to the same separating funnel. The material was repeatedly extracted with 80x3ml, 50x2 ml ethyl acetate (AR Grade) until the ethyl acetate extract became colourless. The combined ethyl acetate extracts were collected and passed over anliydrous sodium sulphate.
  • this material may then be filtered through cotton/filter paper and the aqueous extract transferred into a 250 ml separating funnel.
  • the residue was washed with 10-15 ml of hot water, transferred to the same separating funnel, and then extracted repeatedly with 80 x 3 ml, 50 x 2 ml ethyl acetate (AR Grade) until the ethyl acetate extract became colourless.
  • the combined ethyl acetate extracts were collected and passed over anhydrous sodium sulphate.
  • Extract solution Extracts of Withania, Tinospora and Picrorhiza were added to the required quantities of propylene glycol and purified water and stirred well for 15-20 minutes. This extract solution was filtered through Sparkler Filter Press using Filter Pad and Filter Aid.
  • Wavelength for recording the chromatograms 286 nm (Picroside I) 606 nm (Withaferin A) 636 nm (Cordifolioside A)
  • the plate was developed in the above solvent system over a distance of 8 cm from the point of application. The plate was dried with the help of a hot air-blower.
  • f) Quantification/Calculation For Picroside I content the HPTLC plate was scanned at ⁇ max 286 nm using a deuterium lamp.
  • For Cordifolioside A content the HPTLC plate was sprayed with 10-15 ml of Anisaldehyde-Sulphuric acid reagent and then dried in an oven at a temperature of 105°C for five minutes. The HPTLC plate was scanned at ⁇ max 636 nm using a mercury lamp.
  • the HPTLC plate was sprayed with 10-15 ml of Anisaldehyde-Sulphuric acid reagent and then dried in an oven at temperature of 105°C for five minutes. The HPTLC plate was scanned at ⁇ max 606 nm using a mercury lamp.
  • g) Calculated the content of phytochemical markers in the syrup The percentage content of phytochemical markers in the formulation or syrup was calculated by using the formula x x 100 ASTD D SPL Wherein, A SPL ' Area of test sample corresponding to Withaferin A A STD '• Area of Withaferin A D STD ' Dilution of Reference Standard Solution D SPL ' Dilution of Test sample Table 1 : Content of Phytochemical markers in the syrup

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Abstract

La présente invention porte sur une nouvelle formulation pharmaceutique à base de plantes médicinales qui comprend des extraits de Withania, Tinospora et Picrorhiza ou un mélange d'ingrédients actifs qui ont été extraits de ces plantes, sur un procédé de préparation de la formulation pharmaceutique et sur un procédé d'isolation, d'identification, de quantification ou d'estimation de substances phytochimiques individuelles provenant séparément des plantes respectives et contenues dans la formulation pharmaceutique.
PCT/IB2005/000133 2004-01-19 2005-01-19 Formulation a base de plantes medicinales comprenant des extraits de withania, tinospora and picrorhiza servant de tonique en pediatrie WO2005077392A1 (fr)

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Cited By (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN105017355A (zh) * 2015-07-21 2015-11-04 济南同生生物医药科技有限公司 6’-o-反式桂皮酰梓醇的正相加压柱层析制备方法
CN105111257A (zh) * 2015-09-19 2015-12-02 济南同生生物医药科技有限公司 一种同时制备葡糖乙酰香草苷、10-香荚酰梓醇与6’-o-反式桂皮酰梓醇的工艺方法
WO2018142429A1 (fr) * 2017-02-06 2018-08-09 Muniyal Ayurvedic Research Centre Formulation herbo-minérale pour le traitement du cancer et procédé de préparation associée

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Cited By (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN105017355A (zh) * 2015-07-21 2015-11-04 济南同生生物医药科技有限公司 6’-o-反式桂皮酰梓醇的正相加压柱层析制备方法
CN105111257A (zh) * 2015-09-19 2015-12-02 济南同生生物医药科技有限公司 一种同时制备葡糖乙酰香草苷、10-香荚酰梓醇与6’-o-反式桂皮酰梓醇的工艺方法
WO2018142429A1 (fr) * 2017-02-06 2018-08-09 Muniyal Ayurvedic Research Centre Formulation herbo-minérale pour le traitement du cancer et procédé de préparation associée

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