Review Article

An overview on perforatum Linn.

U M Rao Vattikuti and Veeresham Ciddi* Medicinal Biotechnology Laboratory University College of Pharmaceutical Sciences Kakatiya University Warangal-506 009, Andhra Pradesh, *Correspondent author; E-mail: [email protected] Received 14 September 2004; Revised 2 May 2005

Abstract flowers contain a group of reddish fluorescent dianthrone pigments with 2 Population of the modern world is suffering from dangerous ailments like cancer, AIDS biological activity . It was also reported and depression which have been successfully treated by some herbal medicines. The potentiality of that the biosynthesis of hypericins is Linn. has been proven in treating these diseases. Hypericin, hyperforin connected with morphogenesis and and their analogs are the phytoconstituents of this plant having therapeutic value. The significance formation of dark red coloured oil glands of these constituents is attributed to their botanical safety and therapeutic efficacy. This review on the leaves of the plant3. It has been discusses the pharmacology, extraction and analytical methods of chemical constituents reported on H. perforatum and its plant tissue cultures. known since Greek and Roman times, Dioscorides (1st century A.D) and Galen Keywords: St. John’s Wort, Hypericin, Hyperforin, Biosynthesis, Plant tissue culture, Analysis. described its medicinal properties (2nd IPC code; Int. cl.7 ⎯ A01G 7/00, A61K 35/78, A61P 25/24, A61P 31/12, A61P 31/98, century A.D). It was widely used in the A61P 35/00 folk medicine in European countries as a soothing agent, an antiphlogistic in Introduction approximately one metre high with inflammation of bronchi and urogenital opposite and paired branches. The leaves track, in haemorrhoid treatment, a The genus Hypericum Linn. are opposite and sessile, upto 2cm long, healing agent in the treatment of traumas, consists of nearly 288 species. Of which oblong and contain numerous translucent burns, scabs and ulcers of various kinds H. perforatum Linn., H. maculatum glandular dots, which are visible against and other local and general illness. Today Crantz, H. tomentosum Linn., H. light. The yellow flowers contain 5 petals this drug is little used for these traditional bithynicum Boiss., H. glandulosum with many stamens protruding. These purposes but is largely used in the Gilib. and H. beliaricum Linn. are treatment of depression and reported to contain hypericin1. H. AIDS. perforatum, commonly known as St. John’s Wort (Hindi ⎯ Balsana, Biosynthesis of Bassant, Dendhu) is a potent medicinal Hypericin and plant and is one of the top selling herbs Hyperforin in USA. It is a herbaceous perennial plant of family widely The biosynthesis of distributed in temperate regions of hypericin (Fig 1&2) involves a Europe, Asia, North Africa and USA. In polyketide derivative through 107 India it grows in Himalayas at higher acetate pathway . The altitudes and in the hills of central parts polyketide (1) with a series of intermediates transforms in to of the country. The plant grows Hypericum perforatum

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Chemical composition shown a significant antidepressant activity by inhibiting the enzyme monoamino The plant is reported to contain following categories of chemical constituents: oxidase (MAO)21. The other mechanisms of antidepressant activity are, inhibition Dianthrone derivatives : Hypericin2,4 of dopamine β-hydroxylase22 in vitro, Pseudohypericin5, 6 inhibition of synaptic uptake of serotonin Protohypericin7 and dopamine23, 24, inhibition of catechol- O-methyl transferase25 in vitro, Phloroglucinol derivatives : Hyperforin8 Furohyperforin9 suppression of interlukin-6 in blood Adhyperforin10 samples26 in vivo, modulation of Xanthones11 expression of serotonin receptors27. The antidepressant activity of hyperforin is Essential oil attributed to its inhibition of neuronal Monoterpenes12 : α-Pinene, β-Pinene, Limonene Sesquiterpenes13 : Caryophyllene, Humulene uptake of serotonin, norepinephrine and dopamine like many other antidepressants Flavonols : Catechins14 and also inhibits GABA and α-glutamate uptake28. The antidepressant activity of 15 Flavonoids : Hyperoside hypericum is not only limited to hypericin Quercetin16 Quercetrin17 and hyperforin, xanthones of the plant are Rutin16 also reported to exhibit this property11. Biapigenin18 Antiviral activity ⎯ Kaempferol19 Hypericin is a well-known photosensitizing agent used in the n-Alkanols20 : 0.42% of total herb 1-Tetracosanol (9.7%), 1-Hexacosanol (27.4%), photodynamic therapy of cancer and viral 1-Octacosanol (39.4%), 1-Triacontanol (23.4%) infections. The photodynamic therapy involves the combination of photosensitizing agent and visible light at atrochrysone (2). Atrochrysone on CoA is subsequently lengthened by 3 the absorption wavelength of the dehydration converts into emodine malonyl CoA units and cyclization in to compound. Lavie et al29 demonstrated the anthrone (3). Emodine anthrone readily an intermediate, which would then lead inhibitory effect of hypericin and transforms into hypericin (4) via oxidative to the acylphloroglucinol. Elaboration of pseudohypericin against vesicular free radical chemistry. hyperforin (5) from the unsubstituted stomatitis, influenza virus and herpes The biosynthesis of hyperforin acylphloroglucinol precursor is assumed simplex virus types II and I. Inactivation (Fig 1 & 3) involves isoprenoid moieties, to be with the participation of the geranyl of murine cytomegalo virus, sindbis virus which are derived predominantly via the pyrophosphate and three dimethyl- and HIV-I on exposure to fluorescent light non-mevalonate pathway85. The allylpyrophosphate units. While the with hypericin treatment was reported first acylphloroglucinol (4) moiety is sequence of remaining steps remain to be time by Hudson et al30 and Lopez-Bazzocchi generated via a polyketide type a large extent unidentified. et al31. Both hypericin and pseudohypericin mechanism. This acylphloroglucinol is prevents the viral fusion by the generation formed from isobutyryl-CoA (3) as a starter Pharmacological activities of singlet oxygen up on illumination32. The point, which in turn is formed from two antiviral activity against retroviruses like HIV units of pyruvate via α-acetolactate (1) Antidepressant activity ⎯ involves combination of its photodynamic and α-ketoisovalerate (2). The isobutyryl- Hypericin in a standardized extract has and lipophilic properties.

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Fig. 1 : Structures of Hypericin and Hyperforin

Fig. 2 : Biosynthesis of Hypericin

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c

Fig. 3 : Biosynthesis of Hyperforin

Hypericin binds the cell membranes and significance of hypericin in photodynamic activity. Hyperforin activates a crosslinks the viral proteins leading to its therapy of cancer both in vitro and in mitochondria mediated apoptosis when inability to retrieve the reverse vivo34. Hypericin seems to inhibit EGF added to MT-450 cells. In in vivo, transcriptase activity33. (Epidermal Growth Factor) receptor and hyperforin inhibited the growth of Anticancer activity ⎯ PTK (Protein Tyrosine Kinase) activity. A antologous MT-450 breast carcinoma in Hypericins have been found active against review35 on the mechanism of action of immuno-competent Wistar rats to a similar human leukaemia, squamos cell hypericin and its interaction with cellular extent as that of Paclitaxel without any carcinoma, nasopharyngial carcinoma, components reveals that the signs of acute toxicity36. mouse mammary carcinoma and photogenerated pH drop can achieve the The pharmacology of this plant fibroblasts. Several studies have proven the potentiation of hypericin’s photodynamic also reveals a broad spectrum of activities

Vol 4(5) September-October 2005 371 Review Article like analgesic37, anti-anxiety38, anti- permeabilization, immobilization and contrast to this reduced levels of cellular alcoholic39, antispasmodic40, antioxidant41, various other strategies to improve the capacity to produce hypericin in St. John’s calcium channel blocker42, gene production of active constituents Wort seedlings grown in Nickel expression induction43, wound healing44, compared to the intact plant. This supplemented medium was reported by smooth muscle relaxant45, sleep technique has already been Murch et al 63. potentiation46, anti-inflammatory47 and commercialized for the production of In order to eliminate the quality anti-microbial 48, 49. taxol, shikonin and rosmarinic acid55. impairment in St John’s Wort products, Saxena et al 64 have reported six different Production of Hypericin and Callus formation and plant culture systems for in vitro propagation Hyperforin through tissue cultures regeneration of the plant to compare the biomass and secondary metabolite production with Over the last few years, St. John’s Santarean and Pretto56 reported green houseplants. Of the six culture Wort producers have enjoyed a callus formation and plant regeneration systems they found that growth in large tremendous surge in interest and sales due within few months, from leaf explants of vessel with gelled medium under forced to its potential in the treatment of the species. Li et al 57 reported callus and ventilation (LFV system) was optimal for depression and AIDS. Linde50 revealed the suspension cultures from the leaves and the production of biomass and secondary efficacy of St. John’s Wort when compared stems. Rani et al58 established callus metabolites. Similarly Murch et al 65 to the traditional antidepressants in his cultures of H. perforatum and reported reported the potential for acclimatization clinical studies. Apart from the efficacy the production of hypericin in callus is of in vitro grown St. John’s Wort plantlets in relieving depressive symptoms, St. significantly less than that of intact plant. to a nutrient film technique (NFT) John’s Wort popularity is rapidly They also reported micropropagation hydroponic system in a controlled expanding because of the botanical safety from seeds59. Baruah et al 60 reported in environment green house. The quantities compared to traditional antidepressant vitro regeneration by shoot tip culture. of hypericins and hyperforin were found medications 51-53. Regeneration potentiality was found to to be equal or more than that of green Increasing demand for St. John’s be constant through out the year in house grown . Wort formulations, limited natural sources long-term cultures. The rooted plants coupled with low content of could be successfully transferred to green Cell suspension cultures of H. therapeutically active constituents like house condition. Cordoso and Oliveira61 perforatum hypericin and hyperforin, variation of demonstrated plant regeneration these active constituents in wild plants and from seeds and callus induction from nodal In tissue culture techniques of difficulty in synthesizing hypericins segments of H. brasiliense Choisy. medicinal plants, cell suspension cultures (because of its tautometric forms)54 are Usually secondary metabolites have wide range of application to study the prime reasons to look for the formed in plants serve as defense and optimize the production of important alternative production of these medicinal chemicals not only against predators, secondary metabolites using various agents. One of such alternatives is microbes and competing plants but also strategies. Of the reports on systematic cultivation under controlled to protect the plant survival and growth in H. perforatum, Bias et al 74 described conditions, which provides cultivation of adverse conditions including the presence an in vitro production of hypericins. They the plant with moderate expenditure for of toxic metals. In a way to see the found that the cell aggregate size as an labour and energy sources. In these environmental effects on the biosynthesis important requirement for large-scale circumstances plant tissue culture of hypericins, Briskin and Gawienowski62 production. Kirakosyan and Kaufman75 in technique that is a bit expensive but allows reported that a higher light intensity and their experiment to optimize the hypericin the biotechnologists to attempt lower nitrogen resulted with an increased production in cell suspension cultures micropropagation, elicitation, hypericin and pseudohypericin content. In carried out the fractionation of

372 Natural Product Radiance Review Article heterogeneous cell suspension cultures of fluorescent pigment in the leaves of developed from excised apical buds when the species by centrifugation through differentiated plant from shoot organ compared to the in vivo cultivated plants. sucrose step gradient. The maximum cultures. The production of hypericin, hypericin content ca.0.23mg/g dry wt pseudohypericin and flavonoids is Elicitation was reported from the population with the reported by Kartnig et al1 in largest cells, further fractionation of the H. perforatum, H. maculatum, Elicitors are the agents of biotic cells with the highest floating density H. tomentosum, H. bithynicum, or abiotic origin that can stimulate the revealed the highest percentage of H. glandulosum and H. beliaricum. defense mechanism of the plant cells to hypericin ca. 0.90mg/g dry wt. But the The cell cultures of some strains of induce the excessive production of draw back of this technique is that H. perforatum and H. maculatum secondary metabolites. Plant tissue the cultures are impossible to subculture produced high quantities of hypericin and cultures are the best tools to have the to maintain their growth and production. pseudohypericin. biochemical manipulations with these These reports reveal that the Ishiguro et al 66, 67 reported a new elicitors to enhance the production of optimization of cell aggregates and their xanthone named paxanthonin and active constituents. Enhancement of size are essential to enhance the demethyl paxanthonin together with the production of hypericins and hyperforin production of hypericins in cell known compounds padiaxanthone and was observed when tissue cultures of suspension cultures. In another study tripteroide from callus tissues of H. H. perforatum were subjected to Gabriella Pasqua et al76 reported the perforatum flowers. Schmidt et al 68 elicitation. Kirakosyan et al77 reported that absence of hypericin in callus grown in reported xanthone 6-hydroxylase from mannan at 0.1 mg/ml concentrations dark and suspension cultures derived cell cultures of H. androsaemum Linn. stimulated the production of hypericin and from it. Where as the callus that is used which is capable of hydrolysing 1,3,5 and pseudohypericin 2 and 4 times, for shoot regeneration is grown under a 1,3,7-trihydroxy xanthone at 6th position. respectively when compared to control photoperiod of 16 hours light has They also reported prenylated xanthone shoot cultures. The compound β-1,3- produced dark coloured globules and aglycones and their glycosides from cell glucon stimulated pseudohypericin production of hypericin was observed. cultures of the same plant69. Two new production about 2.5 times when Based on this they conclude that the xanthone glycosides, patuloside A and B compared to control shoot cultures. In a enzymes required for anthranol synthesis have been isolated by Ishiguro et al 70 from similar study on shoot cultures, they are missing in callus and suspension cell suspension cultures of H. patulum reported78 that cork pieces enhanced the cultures. And on other hand cell Thunb. and their structures were production of pseudohypericin by three- differentiation with advanced stage of elucidated by spectral studies. fold (0.4 mg/g dry wt). The stimulating growth is required to obtain the Ishiguro et al71 demonstrated effect of cork tissue was attributed to its biosynthesis of hypericins that in turn is that cell suspension cultures derived from insoluble components either by “massage related with the formation of dark the callus of H. perforatum flowers are effect” or “flotation effect”. Walker et al79 coloured secretary globules. capable of producing a new reported that jasmonic acid improved the phloroglucinol derivative named as growth of biomass and production of Chemical constituents of tissue paglucinol. Yazaki and Okuda72 reported hypericin by two times that of control cell cultured plants the production of procyanidins in H. suspension cultures. They also erectum Thunb. callus and multiple demonstrated the difference in growth and Initiation of shoot organ cultures shoot cultures. This is the first case of hypericin production of cells under light from the germinated seedlings (after tannin production in tissue cultures of this and dark conditions. Increased levels of removal of rootlets) was reported by genus. Guedes et al73 reported the hypericin and hyperforin by abiotic Zdunek and Alfermann3 they found the variation in essential oil components in elicitors, methyl jasmonate and salicylic accumulation of hypericin as a reddish in vitro shoots of H. androsaemum acid in meristem cultures and increased

Vol 4(5) September-October 2005 373 Review Article levels of hypericin by biotic elicitor powder86 and St. John’s Wort powder87. et al 100 reported an extraction procedure Colletotrichum gloeosporioides was The Soxhlet extraction with methanol was with ethyl acetate for the determination reported by Sirvent and Gibson80 in green described for the plant material6,88, 89 and of hypericin in HIV infected plasma house grown plant. commercially available formulations of St. samples. Bauer et al 101 in their work for Understanding the regulation of John’s Wort90. Reported extraction the determination of hyperforin, hypericin biosynthesis, optimization of culture methods with combination of solvents and pseudohypericin in human plasma conditions, selection of cell lines and includes acetone, ethanol and methanol hypericin was extracted from plasma use application of yield improvement (1:1:1) for freeze dried cells91, ethanol samples with phosphate buffer (pH 4.0) strategies are essential to use the tissue and acetone (3:2) for capsule powder92, and acetonitrile mixture (2:3). In the cultures to produce hypericins and methanol and acetone (1:1) for air dried same study, hyperforin was extracted hyperforin on large scale. blossoms by soxhlet extraction93. Michael94 with a mixture containing n-hexane: carried out a similar extraction with ethyl acetate (90:10) at room Extraction methanol and 100mM triethylamine temperature. acetate for commercial St. John’s Wort In modern methods, Barnes102 As the compound hypericin is powder and the extract was directly reported a computer controlled, counter highly photosensitive, the extraction injected in to HPLC system for analysis. current, solvent extraction system using process for St. John’s Wort must be Hansen et al 95 used 80% d-methanol methanol/water solvent in which carried out under dark conditions. (CD3OD) in D2O for extraction followed 99% of the methanol is recovered. Wagner and Bladt2 extracted the plant by HPLC-High Field NMR and Mass Similarly Yanyan and Ang103 reported a material with different solvents and the spectrometry for the structure elucidation small-scale supercritical fluid extraction yields were analyzed for each kind of of naphthodianthrones, flavonoids and (SFE) method for the selective solvent, its concentration and extraction other constituents of H. perforatum extraction of phloroglucinols from St. temperature. They found that optimal crude extract. Sirvent et al96 and Sirvent John’s Wort leaf/flower mixtures using yields were obtained with 80% methanol and Gibson97 reported an extraction supercritical carbon dioxide and at 80°C. Water and ethanol as (40:60), method from fresh plant material for the optimized the conditions. Using this (20 : 80) for the extraction of dried analysis of hypericins involves the removal method hyperforin and adhyperforin were flowers10 and dried hyperici herba81, of chlorophyll with chloroform in the extracted. respectively. Chatterjee et al 82 described absence of light followed by extraction of hydroalcoholic extraction using the remaining plant material with acetone. Analysis methanol and ethanol, it was concluded The dried stem and reproductive that both the solvents produce similar materials were extracted directly with There are many reports for the levels of hypericin but widely acetone. identification and quantification of differing amounts of hyperforin. They also For the extraction of hypericin active constituents viz., hypericin, reported an extraction technique using and hyperforin from cell cultures of hyperforin and their analogs. TLC and

CO2 by which 35% hyperforin was Hypericum spp., fresh or freeze-dried HPLC analysis used by many researchers extracted but the amount of hypericin was cells are subjected to methanolic reported in the literature are summarized. undetectable. extractions with slight modifications using The most commonly followed sonication or shaker1,77,79,98. In in vitro Thin Layer Chromatography method involves the use of methanolic studies, Yanyan et al99 demonstrated extraction in water bath shaker or an extraction with cold acetonitrile for Wagner and Bladt104 described ultrasonic bath. This basic method was the determination of hyperforin in TLC detection of hypericin from followed for the dried flowering tops83, human plasma. In the quantitation of methanolic extracts and commercial trade leaves84, plant material85, mixed capsule hypericin in biological fluids, Liebes samples of H. perforatum on TLC

374 Natural Product Radiance Review Article plates. The solvent system consists of commercial formulations and in vitro detection of hyperforins is at 270 nm and ethylacetate-formic acid-glacialacetic acid- animal studies. hypericins at 590 nm including their water (100:11:11:26). The hypericins protoforms. Brolis et al 83 (1998) were seen as prominent red-violet Dry plant Material described a HPLC method for the fluorescent zones in UV-365nm after identification and quantification of active treatment with NP/PEG (Natural Products- There are few reports of HPLC for constituents using 201 TP 54 RP-18 Poly Ethylene Glycol) reagent. The the estimation of active constituents from column 250 × 4.5mm I.D. 5µm, 300 A°, hypericins were also identified in visible dried powdered plant material. Ganzera protected with an Alltech direct-connect region as green-brown after chemical et al 105 reported a HPLC method for the universal column prefilter of 2µm treatment with 10% pyridine in ethanol. determination of active constituents which porosity. The separation followed a linear Mulinacci et al81 described TLC- consists of a Phenomenex Synergi-NAX RP gradient programme with eluents A-water: densitometry for the quantitative analysis 80 A column (150 × 4.6mm, 4µm). The 85%, phosphoric acid (99.7:0.3 v/v), of hypericin using CAMAG TLC system. The mobile phase consisted of 10µm B-acetonitrile, and C-methanol. The active mobile phase used was toluene- ammonium acetate buffer adjusted to pH constituents were detected at 270nm. ethylacetate-formic acid (50:40:10). 5.0 with glacial acetic acid (A) and a 9:1 Piperopoulos et al93 reported the Densitometric evaluation of the spots was mixture of acetonitrile and methanol (B) determination of naphthodianthrones by performed by using CAMAG TLC Scanner with gradient elution. The detection LC-electrospray mass spectrometry, using II, detection by fluorescence is under an wavelength was 270nm. Sirvent and a Lichrosorb RP 18 column (125 × 4mm excitation wavelength of 313 nm. Kartnig Gibson97 reported a rapid isocratic HPLC i.d., 5µm) protected with a Lichrospher and Gobel84 explained TLC-densitometry analysis of hypericins using Diazem-phenyl 100 RP 18 guard column (4 × 4mm i.d., for the determination of hypericins using (Metachem, 5µm, 250 × 4.6 mm) 5µm). The solvent system was methanol- TLC aluminum sheets coated with silica column, and acetonitrile-methanol-water- acetonitrile (5:4) (solvent A) and 0.1M gel 60 and were developed in unsaturated phosphoric acid (48:40:10:2) as mobile aqueous triethylammonium acetate tanks. The mobile phase used was phase. Primary detection of hypericins was (solvent B) in a gradient programme. The toluene-ethylformiate-formic acid (5:4:1) done at 590nm with secondary detection detection was in the visible range at and the detection was made with UV light at 254nm. 590nm. A HPLC-DAD quantitative analysis at 366nm. Adam et al 85 described a TLC of hypericin described by Mulinacci method for detection of hyperforin using Flowering Tops et al 81 consists of 4.6 × 250mm, 5µm a mixture of hexane and ethyl acetate (9:1 Lichrosorb RP 18 column maintained at v/v) as mobile phase. Spraying a mixture In view of major contributions 26°C. Three mobile phase components of anisaldehyde-sulphuric acid-acetic acid of heterodianthrones and phloroglucinols consists of water adjusted to pH 3.2 by

(1:2:100 v/v) did the detection. Hyperforin from flowers, HPLC methods were addition of H3PO4(component A), afforded a blue spot with Rf value of 0.42. established for the extractions exclusively methanol (component B) and CH3CN made from air-dried blossoms. Poutaraud (component C) were used to prepare High Performance Liquid et al 10 reported a HPLC method for the the multistep linear solvent gradient Chromatography quantification of naphthodianthrones and programme. Hypericin and pseudohypericin phloroglucinols using short linear were easily identified and quantified at The pharmaceutical significance gradient system. The analysis was carried 590nm. of St. John’s Wort by the active constituents out at 40oC on a Nucleosil – 100 end Tissue Cultures has led to their evaluation by various capped RP18 column using three solvents isocratic and gradient types of HPLC A-acetonitrile, B-water: 85% phosphoric To study the accumulation of methods in the dried plant material, acid (99.7:0.3, v/v) and C-methanol with phytoconstituents in tissue culture flowering tops, tissue cultures, a linear gradient programme. The experiments, Tolonen et al 91 described

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RP18 Review Article

a HPLC method for determination of fractions from in vivo and in vitro RP-C18 3µm, 250 × 4mm column with a naphthodianthrones and phloroglucinols biomass of Hypericum spp. The analysis linear gradient programme. The mobile from cell culture extracts. The compounds was carried out using a Lichro Cart RP 18 phase consists of A-0.5% TFA in Water, were eluted in Waters Xterra RP18, 2.1 × end capped Supersphere column (150 × B-0.5% TFA in mixture of methanol- 50mm column with 3.5 µm particle size 4mm ID, 4µl) with a precolumn of the acetonitrile (13:7) in linear gradient at 35oC. The elution was done with a same material. Mobile phase consisted of programme. Absorption was measured gradient programme using aqueous water-formic acid (95:5) as eluent A and with PDA detector at full spectrum (200- 20mM ammonium acetate (A) and methanol as eluent B with a gradient 790 nm). Li and Fitzloff87 established an acetonitrile (B). All the naphthodianthrones programme. The detection of hypericin RP-HPLC method with PDA detection for and phloroglucinols were well separated was done at 590nm. the determination of major constituents and detected by tandem mass in St. John’s Wort dietary supplements. The spectrometry. Kirakosyan et al 77 reported Formulations analysis was carried out at 20°C on a a HPLC method for the analysis of Waters YMC OD3-Aq1N RP-18 column, hypericins in shoot cultures using Owing to the huge demand for which was protected by a Waters delta- Cosmosil 5C 18-MS C 4.6 × 250 mm hypericin and hyperforin as PAK RP-18 guard column. The mobile column. The mobile phase consisted of antidepressants and in the treatment of phases consisted of water (containing A-0.5% TFA (Trifluoro Acetic acid) in AIDS, number of St. John’s Wort 20% methanol and 0.5% TFA) and water, B-70% MeCN - 29.5% methanol - formulations are coming in the market. acetonitrile (containing 10% methanol 0.5% TFA in a gradient-time programme. In order to ascertain the quality of these and 0.5% TFA) and the analysis followed The peaks were observed on the products the most sensitive and a linear gradient programme. The UV chromatogram by UV absorption at 588 reproducible methods of HPLC were absorption for hypericin and hyperforin nm. Walker et al79 in a HPLC-MS method reported. along with other constituents were studied for the analysis of hypericin in cell Catharina et al86 described a at 270nm and 590 nm. Michael94 suspension cultures, used a 5µm, C18 HPLC method for the determination of described HPLC analysis of hypericin with column of 25 × 4.6 cm size. The solvent hypericin and pseudohypericin derivatives PDA and MS detection. The HPLC column system used was A-70% solution of 1% in dietary supplements and functional used for system 1 was C8Waters, ammonium phosphate (pH 7 adjusted foods. The constituents were eluted in a 150 × 2.1 mm with 0.3 ml/min flow with NaOH) and 30% acetonitrile, B- reverse phase column, Phenomenex Luna rate and for system 2 was C8 Waters acetonitrile, 30% water with a gradient 3µm, C18(ODS-2) 4.6 × 150 mm using 150 × 3.0 mm with 0.4ml/min flow rate. programme. The hypericin was detected acetonitrile-triethylammonium acetate The chromatographic eluents were A- in visible absorbance at 590 nm by a PDA buffer (4:1) as solvent system. 10mM triethylamine acetate (pH 7.0), B- (Photo Diode Array) detector. Kartnig et Benzo[k]fluoroanthene was used as methanol, C-acetonitrile in a linear al 1 described a HPLC for the internal standard. The components were gradient programme. The detection of determination of hypericins in cell detected at 290nm and 590nm. hypericin was done at 588 nm. Hansen cultures of various Hypericum species Confirmation analysis was carried out with et al95 in a HPLC method on-line and their chemotypes. The analysis was LC/electrospray ionization (ESI)/MS coupled to High-Field NMR and mass carried out with a column of Lichro Cart analysis. spectrometry for structure elucidation of RP 18 Supersphere 250 × 4mm with RP 8 Liu et al 92 established a RP-HPLC naphthodianthrones, flavonoids and precolumn. Eluent used was acetonitrile- method with PDA detection and LC- other constituents of H. perforatum methanol-water-phosphoric acid electrospray ionization - MS confirmation used Knauer column 120 × 4 mm (55:20:24:1). The UV detection was done of major active constituents in St. John’s i.d. packed with Apex-1 ODS, 5µm at 254 nm. Dias et al 98 reported a HPLC Wort dietary supplements. The analysis column packing material. The mobile method for the analysis of the phenolic was carried on a Lichrospher end-capped phase was made up of A-acetonitrile and

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0.1% acetic acid (5:95 v/v), B-acetonitrile phase consists of methanol-acetonitrile hyperforin, samples were analyzed at and 2mM ammonium acetate (98.5 v/v) (3:2 v/v) mixed with water (92:8). The 50°C on a Phenomenex Luna C18 column in gradient conditions. The detection of hyperforin was detected at 287 nm with (5µm, 250 × 4.6mm i.d.) with a guard components was done at 254nm. Denke Waters model 996 PDA detector. column and quantified by UV detection et al 88 used Hypersil ODS 5µm, Leibes et al 100 reported a HPLC at 273nm. The mobile phase 250 × 4.6 mm column for the analysis of method for the quantification of hypericin consisted of acetonitrile, 0.01M sodium hypericin in their studies on biochemical in biological fluids. The analysis was hydrogen phosphate buffer (pH 2.4) activities of extracts. The mobile phase carried out on a reverse phase, phenyl (90:10). consisted of mixture of methanol/ethyl 4µm, and 0.45×10cm radial pack acetate/buffer, pH 2.1 (800/195/205/v/v/ analytical column. The mobile phase Conclusion v) with isocratic elution. Hypericins were consisted of Solution A-70% solution of detected at 590nm. In another report90 for 0.1% ammonium phosphate (pH 7 with The review describes chemical the analysis of constituents they used NaOH) and 30% acetonitrile, Solution B- profile, pharmacology and analytical Macherey – Nagel EC 125/4.6 Nucleosil 70% acetonitrile and 30% water, in a methods of H. perforatum with special

100-5 C18 AB column. The eluents were A linear gradient programme. The detection reference to hypericin and hyperforin. - 50 mmol/L sodium phosphate (pH 2.3), of hypericin in visible range was done at Analysis by TLC and a number of HPLC B-50 mmol/L sodium phosphate, (pH 590nm. Chi and Franklin106 reported a methods were reported for the 2.3), 40% methanol and 49% acetonitrile HPLC method for the measurement of determination of hypericin and hyperforin in gradient-time programme. The hypericin in plasma using 5µm particle from plant material, tissue cultures, detection of constituents was done at size mixed mode (C18/CN) analytical commercial products and in vitro 254nm using photodiode array detector. column and a model 975 UV detector set (plasma) studies have been summarized Reyes and Koda5 described a HPLC method to the optimal wavelength of 272nm using for further applications. Limited natural for the simultaneous determination of end point detection. The mobile phase sources, variation of amounts of active hypericins and stabilized hyperforin consisted of 81% acetonitrile in water constituents in wild plants, efficacy and in commercial St. John’s Wort adjusted to pH 4.5 (with 1M H3PO4). safety of natural phytoconstituents led to 101 preparations using C18 column. Isocratic Bauer et al reported a HPLC method their alternative production by in vitro elution was done with a mobile phase with fluorescence and UV detection for methods. Hence, tissue culture methods consisting of acetonitrile and 0.3% v/v the determination of hypericin, have been described for the production phosphoric acid (90:10 v/v), with pseudohypericin and hyperforin in human of these valuable secondary metabolites. simultaneous fluorescence (315/590 nm, plasma. Hypericin and pseudohypericin To meet the increasing demand excitation/emission) and UV (273 nm) were separated at 60°C in a Lichrospher for novel compounds, reports on detection. RP select B column (5µm, 250 × 4.6mm micropropagation and elicitation of key i.d.) with a guard column and constituents by tissue culture methods In vitro Animal Studies quantification by fluorescence detection could offer a possible solution. To further at 315/590nm. For mobile phase strengthen these approaches there is HPLC method of analysis was preparation 6.1ml of concentrated a need to attempt transformed root even successful in estimating the phosphoric acid in 950ml distilled water, cultures, permeabilization and concentration of active constituents in pH adjusted to 4.0 by NaOH (30% in immobilization in cell cultures. in vitro studies (human plasma). Yanyan water) made up to volume 1000ml with Molecular studies and metabolic et al 99 in a HPLC method reported the distilled water. A 300ml of this aqueous engineering of hypericin, hyperforin and determination of hyperforin in human solution was combined with 450ml their analogues may contribute plasma using Luna C18-150 × 4.6 mm, methanol and 250ml tetrahydrofuran to significantly to enhance their production 3µm particle size column. The mobile constitute the mobile phase. For through tissue cultures.

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St. John's Wort may be toxic during pregnancy and lactation in rats In recent years, concerns about St. John's Wort (Hypericum perforatum Linn.)use during pregnancy and breastfeeding have emerged. The scientists from Italy investigated the toxicity of this herb in Wistar rats, administered prenatally and during breastfeeding (from 2 weeks before mating to 21 days after delivery). Two doses of the extract were chosen, 100 mg/kg per day, which, based on surface area, is comparable to the dose administered to humans, and 1000 mg/kg per day. A microscopical analysis of livers, kidneys, hearts, lungs, brains, and small bowels was performed. A severe damage was observed in the livers and kidneys of animals euthanized postnatally on days 0 and 21. The lesions were more severe with the higher dose and in animals that were breastfed for 21 days; however, an important renal and hepatic damage was evident also with the dose of 100 mg/kg per day. In addition, similar serious hepatic and renal lesions were evident also in animals that were exposed to hypericum only during breastfeeding. The results indicated that appropriate histological studies should be performed in other animal species to better evaluate the safety of hypericum extracts taken during pregnancy and breastfeeding [Gregoretti B, Stebel M, Candussio L, Crivellato E, Bartoli F and Decorti G, Toxicity of Hypericum perforatum (St. John's Wort) administered during pregnancy and lactation in rats, Toxicol Appl Pharmacol, 2004, 200(3), 201-205].

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