Pharmaceutical Sciences, March 2015, 20, 135-140

http://journals.tbzmed.ac.ir/PHARM

Phytochemical Analysis of Danae Racemosa L. Moench Leaves Fatemeh Fathiazad*1, Sanaz Hamedeyazdan1,2 1Department of Pharmacognosy, Faculty of Pharmacy, Tabriz University of Medical Sciences, Iran. 2Student’s Research Committee, Department of Pharmacognosy, Faculty of Pharmacy, Tabriz University of Medical Sciences, Iran.

A B S T R A C T A R T I C L E I N F O

Article Type: Background: Danae racemosa (L.) Moench (Ruscus racemosa L., Original Research ) is an erect, muchbranched evergreen shrub, native in the mountains from Syria to Iran, commonly used for its decorative green foliage Article History: in fresh flower arrangements. Despite its importance, far too little attention Received: 28 June 2014 Accepted: 8 November 2014 has been paid to gather detailed documentations of chemical constituent present in extract of D. racemosa. Methods: In this study, leaves of D. racemosa were collected from north of Iran, next, extracted via maceration Keywords: Danae racemosa followed by partitioning through solvents of different polarity. The Asparagaceae qualitative–quantitative chemical composition of the extract was determined Flavonoid using high-performance liquid chromatography. Results: Two major UV-visible shift reagents flavonoids, quercetin and kaempferol, were separated whilst their identities Quercetin 1 Kaempferol were confirmed by UV-visible, shift reagents and H-NMR spectroscopy.

Introduction In a time of increased considerations to natural erect, much branched evergreen shrub growing products as potential safe remedies, it is as under the shade of forest trees, with green shoots important as ever to gather detailed and glossy leaves along with thick unarmed documentations of the latter in different aspects. alternate cladophylla and terminal racemese of Accordingly, natural resources, particularly white-yellow flowers followed by red berries is higher species, continue to be important frequently used for its decorative green foliage in sources of natural medicines which represent a fresh flower arrangements.4 Although there have challenge to science due to various aspects, been a great number of studies referring the including their chemical constituents. beneficial of containing biologically active Danae racemosa (L.) Moench (Syn.; Ruscus chemical constituents, far too little attention has racemosus L.) ("Hamishak" in Persian and been paid to gather detailed documentations of "Alexandrian Laurel" in English) belongs to the chemical constituents present in extract of D. family Asparagaceae.1 The family Asparagaceae racemosa. Hence, the present study was is in the major group Angiosperms (Flowering conducted to detect some biologically active plants) that is placed in the order of chemical constituents, naturally occurring in the monocots. The most complete revision of the extract of the plant. This survey is an ongoing family was Baker’s classification (1875), who point in our study on the plants of Iranian flora. divided the natural order Liliaceae into three Regarding our previously published papers on the suborders; Liliaceae proper, Colchicaceae and elicited systemic dose-related antinociceptive Asparagaceae, based on the fruit and stamen properties of D. racemosa extracts on both characteristics.2 The Asparagaceae was chemical and thermal nociceptive models6 and characterized by baccate fruit and introsed spermatogenesis in ,7 herein we report on the stamens and then subdivided globally into 143 extraction, isolation and structural elucidation of genera and over 3500 species.3 D. racemosa two flavonoids from the leaves of D. racemosa. member of family Asparagaceae is native in the mountains from Syria to Iran and Transcaucasia.4 Materials and Methods The plant naturally grows in higher lands mainly General experimental procedures in the north parts of Iran including Guilan, All solvents in the present work were purchased Mazandaran and Golestan provinces that are from Merck (Germany). HPLC separation was located in the south of the Caspian Sea and north performed in a Shimadzu prep-HPLC System of the Elburz mountains on latitude of N35°50', coupled with UV detector. A Shim-Pak CLC – 38°30' and longitude E48°30', 56°10' and ranges RP18 preparative high performance column of 700-1600 mm precipitation.5 D. racemosa, an chromatography (HPLC) column (10 µm, i.d.:

*Corresponding Author: Fatemeh Fathiazad: Department of Pharmacognosy, Faculty of Pharmacy, Tabriz University of Medical Sciences, Iran. Tel: +98 41 36692405, Fax: +98 41 33344798, E-mail: [email protected]

Analysis of Danae Racemosa L

250 mm × 20 mm; mobile phase: 0-80 min, defatted powdered aerial parts were extracted acetonitrile - distilled water (40-60) as the eluent; with 70% methanol in water (2L) by maceration flow-rate: 6 ml/min, injection volume: 0.5 ml and at room temperature. The methanol of resultant detection at 254 nm was used in the hydroalcoholic extract was evaporated at 40 ◦C chromatography. UV-visible spectrum of each under reduced pressure, affording the aqueous compound was determined using a Shimadzu residue. Subsequently, the aqueous residue was UV-2100A spectrometer in methanol and after fractionated with EtOAc and n-butanol addition of different shift reagents such as respectively. According to the TLC analysis of NaOMe, AlCl3, ALCl3/HCl, NaOAc and the fractions on analytical silica gel GF254 plate NaOAc/H3BO4 at 190-500 nm. H-NMR spectra using a mixture of n-hexane-EtOAc-MeOH were recorded in MeOH-d4 on a Bruker DRX (6:3:1) as eluent and 5% AlCl3 reagent for 200 MHz NMR spectrometer. detection, the EtOAc fraction was determined to be rich in flavonoids. As a consequence, the Plant material EtOAc fraction was subjected to preparative The leaves of Danae racemosa (L.) Moench were HPLC providing several fractions. On separation, collected during June–July from Shirgah forests two fractions with retention times of 36.46 and in Mazandaran province in Iran. A voucher 52.11 minutes each containing one major specimen of the plant (Tbz-Fph-751) representing flavonoid (Figure 1), were dried and dissolved in this collection has been retained in the herbarium small volume of methanol and kept in refrigerator of the Faculty of Pharmacy, Tabriz University of at +2°C. The formed crystals were separated by Medical Sciences, Tabriz, Iran. filteration and dried to provide compound 1 and compound 2. The structures of compound 1 Extraction, isolation and structure elucidation of (45mg) and 2 (32mg) were elucidated by flavonoids interpretation and comparison of their spectral The dried aerial parts (200g) were grounded and data (UV and 1H-NMR) with those published defatted by treatment with n-hexan (1L). The references.8,10

Quercetin

Kaempferol

Figure 1. HPLC chromatogram of the flavonoids in EtOAc fraction of D. racemosa

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Results and Discussion tables 1 and 2. The UV spectrum of methanolic Two major flavonoids were detected in EtOAc solution of compound 1 which was separated as fraction of the leaves of D. racemosa, where the yellow crystals exhibited two major absorption structures were deduced by UV-visible and 1H- bands at 371 nm (band-I, cinnamoyl system) and NMR spectral data analyses. The spectroscopic 255 nm (band-II, benzoyl system) (Table 1), data of the separated flavonoids are outlined in which confirmed the flavonol structure.

Table 1. UV-visible absorption peaks of the compounds 1 and 2 in methanol and with various shift reagents.

Compound MeOH NaOMe AlCl3 AlCl3/HCl NaOAc NaOAc/H3BO3

454 424 370 419 (sh) 393 364 (sh) 358 (sh) 386 1 298 (sh) 328 325 (sh) 313 (sh) 300 (sh) 259 254 273 271 264 363 422 422 426 322 (sh) 349 (sh) 349 (sh) 390 370 2 317 (sh) 295 (sh) 300 (sh) 300 (sh) 274 264 280 264 267 267

1 Table 2. H-NMR (300 MHz) data of the compounds 1 and 2 in CD3OD.

Chemical shift (δH) in ppm Chemical shift (δH) in ppm Position 1 (Quercetin) 2 (Kaempferol) 1 - - 2 - - 3 - - 4 - - 5 - - 6 6.16 (d, J = 2.1 Hz) 6.17 (d, J = 2.1 Hz) 7 - - 8 6.37 (d, J = 2.1 Hz) 6.38 (d, J = 2.1 Hz) 1' - - 2' 8.07 (d, J = 2.1 Hz) 8.01 (d, J = 9.0 Hz) 3' - 6.90 (d, J = 9.0 Hz) 4' - - 5' 6.90 (d, J = 8.4 Hz) 6.90 (d, J = 9.0 Hz) 6' 7.62, 7.60 (dd, J = 8.4 , 2.1 Hz) 8.01 (d, J = 9.0 Hz)

The structure of each flavonol compound was like quercetin. The flavonol-aluminum complex verified according to UV absorbance spectra and presents a bathochromic shift corresponding to known shift reagents. In the case of AlCl3 and form a number of complexes. On addition of acid, AlCl3/HCl reagents, aluminum chloride forms a the acid-labile catechol complex will dissociate complex with the hydroxyl groups at C3- or (and) bringing about a hypsochromic shift compared to C-5 and the ketone group at C-4 of the flavonol the aluminum chloride spectrum. Furthuremore, structure and also forms an acid-labile complex NaOAc ionizes only the flavonols with a free 7- with the catechol functional group in a compound hydroxyl exhibiting a Band II shift (5–20 nm).

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Besides, decomposition would occur only if spectrum (Table 2), the data indicated a 5,7- alkali-sensitive hydroxyl patterns are present dihydroxylated pattern for ring A (two meta- (e.g., 3-, 3′-, 4′-). Thus, degeneration of the band I coupled doublets at δH 6.16 and 6.37 ppm, (J = in the presence of NaOMe, indicates free 2.1 Hz) and a 3′,4′-dihydroxylation pattern for 11 hydroxyl groups at C-3 and C-4'. Herein, ring B: ABX system signals at δH 6.90 ppm (1H, hypsochromic shifts with AlCl3/HCl and d) with J = 8.4 Hz attributed to hydrogen atom H- bathochromic shift in bond-I (16 nm) with 5' and δH 7.62 and 7.60 ppm, (1H, dd) with J = NaOAc/H3BO3 supported the presence of 3, 3′, 4′ 8.4, 2.1 Hz for H-6' followed by 8.08 ppm, (1H, trihydroxy system in the compound. d) with J = 2.1 Hz for H-2', allowing the Bathochromic shifts with NaOAc were related to compound to be recognized as quercetin which is 7-hydroxyl and the bathochromic shift with in consistence with the previously published data 1 12-16 AlCl3/HCl to 5-hydroxyl. Regarding the H-NMR for this compound (Figure 2) .

OH 3 ' OH 2 ' OH 2 ' HO 8 O 5 ' 6 ' HO 8 O 5 ' 6 ' 6 OH 6 OH OH O

OH O

Quercetin Kaempferol

Figure 2. Chemical structures of the flavonoids in EtOAc fraction of D. racemosa.

In the case of compound 2, UV absorption The results of quantitative determination by spectrum in methanol exhibited two absorption HPLC of quercetin and kaempferol in D. maximums at 363 nm (band-I, cinnamoyl system) racemosa indicated that the quercetin and and and 264 nm (band-II, benzoyl system) Kaempferol content of D. racemosa leaves were indicating a flavonol compound. Furthermore, 0.6% and 0.8% (based on dry weight of the shifts obtained with diagnostic reagents: NaOMe, leaves), respectively. However, it can be used as 280, 317 (sh), 426; AlCl3: 267, 300 (sh), 349 (sh), an economical source of quercetin and 422; AlCl3/HCl: 267, 300 (sh), 349 (sh), 422; kaempferol in pharmaceutical industry. Formerly NaOAc: 274, 390; and NaOAc/H3BO3: 264, 370 in our published paper on assessment of the D. indicated the presence of some free hydroxyls. racemosa hydroalcoholic extract effect on its Considering the UV spectrum in methanol and its antinociceptive properties we determined marked changes after the addition of the customary shift dose-related antinociception in a number of reagents in comparison with the reported data, as chemical nociception models: formalin-induced it has been stated above, it was suggested that the hindpaw licking, acetic acid-induced abdominal compound 2 might be a flavonol with free constriction, hot-plate and rota-rod tests in rat hydroxyl groups at positions C-5, 7 and C-3 and models.6 We also found that D. racemosa 4′. Additionally, 1H-NMR spectrum of compound hydroalcoholic extract could increase sperm 2 displayed characteristic signals of the count, viability and motility along with increasing kaempferol nucleus: two doublets at δH 6.17 and in serum TAC (total antioxidant capacity) and 6.38 ppm (J = 2.1 Hz), assigned to the H-6 and H- decreasing in serum MDA (malondialdehyde) in 8 protons, respectively and a pair of A2B2 rats. These findings confirm the antinociceptive aromatic system protons at δH 6.90 and 8.01 ppm and inducing spermatogenesis activities of D. (J = 9.0 Hz), assigned to H-3', 5' and H-2', 6', racemosa extract in vitro via antioxidant respectively. Consequently, based on the 1H properties of the isolated flavonoids.21-23 NMR and UV-visible data and also comparison of Likewise, it has been widely accepted that the data given in the literature, the structure of the flavonoids encompass quite a lot of health compound 2 was identified as kaempferol (Figure benefits and diets rich in flavonoids alleviates and 2).17-20 prevents many serious diseases.23-30

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