Home | About PR | Editorial board | Search | Ahead of print | Current Issue | Archives | Instructions | Subscribe | Advertise | Contact us |   Login 
Pharmacognosy Magazine
Search Article 
  
Advanced search 
 


 
 Table of Contents 
ORIGINAL ARTICLE
Year : 2013  |  Volume : 5  |  Issue : 1  |  Page : 17-21  

Flavonoid glycosides and pharmacological activity of Amphilophium paniculatum


1 Department of Chemistry of Natural Compounds, National Research Centre, Dokki, Egypt
2 Department of Pharmacognosy, Faculty of Pharmacy, Cairo University, Dokki, Cairo, Egypt
3 Department of Pharmacognosy, National Research Centre, Dokki, Cairo, Egypt

Date of Submission27-Mar-2012
Date of Decision25-Apr-2012
Date of Web Publication08-Jan-2013

Correspondence Address:
Mahmoud I Nassar
Department of Chemistry of Natural Compounds, National Research Centre, 12622 Dokki,Cairo
Egypt
Login to access the Email id

Source of Support: None, Conflict of Interest: None


DOI: 10.4103/0974-8490.105643

Rights and Permissions
   Abstract 

Background: Nothing is reported on Amphilophium paniculatum (L.) Kunth. This study aimed at investigation of chemical constituents of the leaves of Amphilophium paniculatum, grown in Egypt, in addition to pharmacological evaluation. Materials and Methods: Isolation of a new compound, along with 5 known flavonoids. Pharmacological activities were carried out on different extracts of A. paniculatum leaves. Results: Identification of a new flavone glycoside, acacetin 8-C-β-D- glucopyranosy l-(1→2)-α-L-rhamnopyranoside (1) in addition to 5 known flavonoids. The 70% ethanol crud extract and its successive chloroform, ethyl acetate, and 100% ethanol extracts showed significant anti-inflammatoryactivity,analgesic effect, antipyretic activity, antioxidant activity, and anti-hyperglycemic activity. Determination of the median lethal dose (LD 50 ) revealed that the different extracts were safe.

Keywords: Amphilophium paniculatum, bignoniaceae, flavonoids, pharmacology


How to cite this article:
Nassar MI, Aboutabl ESA, Eskander DM, Grace MH, EL-Khrisy EDA, Sleem AA. Flavonoid glycosides and pharmacological activity of Amphilophium paniculatum. Phcog Res 2013;5:17-21

How to cite this URL:
Nassar MI, Aboutabl ESA, Eskander DM, Grace MH, EL-Khrisy EDA, Sleem AA. Flavonoid glycosides and pharmacological activity of Amphilophium paniculatum. Phcog Res [serial online] 2013 [cited 2020 Aug 12];5:17-21. Available from: http://www.phcogres.com/text.asp?2013/5/1/17/105643


   Introduction Top


Bignoniaceae is a family of flowering plants, which comprises about 800 species in 120 genera, some species are cultivated as ornamentals, distributed in tropical and subtropical of South America, Africa, and India. [1],[2],[3] Nearly all members of this family have woody stems. The genus Amphilophium[4],[5] has woody liana, leaves 2-3 foliolate, and fruits are smooth, flat, and somewhat woody. Nothing is reported on Amphilophium paniculatum (L.) Kunth. This study aimed at investigation of chemical constituents and pharmacological activity of the leaves of Amphilophium paniculatum, grown in Egypt, in order to support the possibility of its uses as a natural resource in therapeutics and to demonstrate the correlation between chemical composition and bioactivity.


   Materials and Methods Top


General

NMR spectra were recorded on a JEOL EX-500 MHz NMR spectrometer in DMSO-d6 using TMS as internal standard, mass spectra (±) ESI-MS: LCQ Advantage Thermo Finnigan spectrometer.

Plant material

Amphilophium paniculatum (L.) Kunth leaves (Family Bignoniaceae) were collected from El-Orman garden, Giza, Egypt. The plant samples were kindly identified by Mm. Tressa Labib, Taxonomist, El-Orman garden, Giza, Egypt.

Extraction and isolation

The air-dried powder of Amphilophium paniculatum leaves (1 kg) was extracted by 70% ethanol. The aliquot of ethanol extract was evaporated under reduced pressure to give 150 g extract, which was suspended in water (1L) and then extracted successively with petroleum ether, chloroform, ethyl acetate, and n-butanol. The successive extracts were separately evaporated under reduced pressure to yield 20 g, 25 g, 30 g, and 20 g respectively. The n-butanol fraction (20 g) was subjected to column chromatography on polyamide 6S. The column was eluted with distilled water followed by water/ethanol step-gradient. Finally, the column was eluted with absolute ethanol to ensure a perfect elution process. The separated fractions resulting from the column were examined by PC using solvent systems (BAW and 15% ACOH) under UV light. The similar fractions were collected together to give 6 major fractions, which were subjected to Sephadex LH-20 columns to give a new flavonoid, acacetin 8-C-β-D-glucopyranosyl-(1→2)-α-L-rhamnopyranoside (1), in addition to 5 known flavonoid compounds.

Animals

Albino mice of 25-30 gm body weight and adult male Albino rats of Sprague Dawely Strain of 130-150 g body weight were used in this study, obtained from the animal house colony of National Research Centre, Egypt. All animals were kept under the same hygienic conditions and on a standard laboratory diet.

Chemicals and kits

Metformin (Chemical Industries Development, Giza, ARE), alloxan (Sigma Co: Cairo, Egypt). Biodiagnostic kit for assessment of blood glucose and glutathione levels, Glutathione kit (Wak Company-Germany) for the assessment of antioxidant activity. Indomethacin (Epico, Egyptian Int. Pharmaceutical Industries Co.). Carrageenan (Sigma Co.) Tramadol (October Pharma, Egypt). Aceticacid (Sigma Co.). Vitamin E (Pharco Pharmaceutical Co.). Paracetamo l (Misr Co., Egypt, Cairo).

Doses of the tested materials were administered orally by gastric tube. [6]

Pharmacological screening

The air-dried powder of Amphilophium paniculatum leaves (600 g) was extracted by 70% ethanol. The aliquot of ethanol extract was evaporated under reduced pressure to give 65 g extract. The residue of extract was suspended in water 1000 ml and then extracted successively with chloroform, ethyl acetate, and 100% ethanol. The extracts were separately evaporated under reduced pressure to yield 14 g, 12 g, and 14 g, respectively. The extracts were kept in tightly sealed sample tubes for the biological study.

Median Lethal Dose (LD 50 )

Determination of the LD 50 of extracts of A. paniculatum leaves was estimated where all doses were expressed in terms of extract weight/animal weight. [7] Preliminary experiments were done to determine the minimal dose that kills all animals (LD 100 ) and the maximal dose that fails to kill any animal. Several doses at equal logarithmic intervals were chosen in between these two doses, each dose was injected in a group of 6 animals by subcutaneous injection. The mice were then observed for 24 hrs., and symptoms of toxicity and mortality rates in each group were recorded, and the LD 50 was calculated.

Anti-inflammatory activity

This effect was determined according to the method described by Winter et al.[8] Sixty male albino rats, weighing 130-150 g were divided into 10 groups, each of 6 animals; first group: Rats that received 1 ml of saline serving as control,second group: Rats that received 50 mg/Kg of 100% ethanol of plant extract, third group: Rats that received 100 mg/Kg of 100% ethanol of plant extract, fourth group: Rats that received 50 mg/Kg of 70% ethanol of plant extract, fifth group: Rats that received 100 mg/ Kg of 70% ethanol of plant extract, sixth group: Rats that received 50 mg/Kg of chloroform of plant extract, seventh group:Rats that received 100 mg/Kg of chloroform of plant extract, eighth group: Rats that received 50 mg/Kg of ethyl acetate of plant extract, ninth group: Rats that received 100 mg/Kg of ethyl acetate of plant extract, tenth group: Rats that received 20 mg/Kg of the reference drug, indomethacin.

One hour later, all the animals received a sub-plantar injection of 0.1 ml of 1% carrageenan solution in saline in the right hind paw and 0.1 ml saline in the left hind paw. Four hours after drug administration, the rats were sacrificed; both hind paw excised and weighed separately.



Analgesic activity

Animals were acclimatized to the laboratory conditions for at least 1 hr. before testing and were used once during the experiment.

Acetic acid induced writhing test: Sixty Swiss male albino mice (20-25 g) were divided into 10 groups, each of 6 animals were used. The first group: Received 1 ml of saline serving as control, second group: Received 50 mg/kg of 100% ethanol of plant extract, third group: Received 100 mg/kg of 100% ethanol of plant extract, fourth group: Received 50 mg/kg of 70% ethanol of plant extract, fifth group: Received 100 mg/kg of 70% ethanol of plant extract, sixth group: Received 50 mg/kg chloroform of plant extract, seventh group: Received 100 mg/kg chloroform of plant extract, eighth group: Received 50 mg/kg of ethyl acetate of plant extract, ninth group: Received 100 mg/kg of ethyl acetate of plant extract, tenth group: Received the reference drug, tramadol 20 mg/kg. Thirty minutes later, 0.6% acetic acid was injected intraperitoneal (0.2 ml /mice). Each mouse was then placed in an individual clear plastic observe chamber, and the total no of writhes/30 min. was counted for each mouse. [9]

Antipyretic activity

This effect was carried out following the method of Buch et al.[10] Thirty-six male albino rats of average body weight 100 g were divided into 6 groups; each group of 6 animals: First group: Rats that received 1 ml of saline serving as control,second group: Rats that received 100 mg/Kg of 100% ethanol of plant extract,third group: Rats that received 100 mg/Kg of 70% ethanol of plant extract, fourth group: Rats that received 100 mg/ Kg of chloroform of plant extract,fifth group: Rats that received 100 mg/Kg of ethyl acetate of plant extract, sixth group: Rats that received 20 mg/kg of the reference drug, paracetamol. The normal rectal temperature was recorded before the start of the experiment. Pyrexia was induced by intramuscular injection of 1 mg/100 g body weight of 44% yeast suspension. The site of injection was then massaged to spread the suspension beneath the skin. After 18 hrs, the rectal temperature was recorded for all groups to serve as the base line of elevated body temperature, to which the anti-pyretic effect will be compared. One and two hrs later, other records of rectal temperature were determined.

Antioxidant activity

Male albino rats of the Sprague Dawely Strain (130 g-140 g) were injected intraperitoneally with alloxan (150 mg/kg body weight) to induce diabetes mellitus. [11] Sixty-six rats were divided into 11 groups each of 6 animals: First group: Normal rats served as negative control received 1 ml saline, second group: Diabetic rats served as positive control received 1 ml saline, third group: Diabetic rats that received 7.5 mg/kg b.wt. of vitamin E as reference drug, fourth group: Diabetic rats that received 50 mg/Kg of 100% ethanol plant extract, fifth group: Diabetic rats that received 100 mg/Kg of 100% ethanol plant extract, sixth group: Diabetic rats that received 50 mg/Kg of 70% ethanol plant extract, seventh group: Diabetic rats that received 100 mg/Kg of 70% ethanol plant extract,eighth group: Diabetic rats that received 50 mg/Kg of chloroform plant extract,ninth group: Diabetic rats that received 100 mg/ Kg of chloroform plant extract, tenth group: Diabetic rats that received 50 mg/Kg of ethyl acetate plant extract, eleventh group: Diabetic rats that received 100 mg/Kg of ethyl acetate plant extract. After 7 days, blood samples were collected from the rats.

Determination of blood glutathione

Glutathione in blood was determined according to the method of Beutler et al.[12]

Glutathione (GSH) concentration in blood = A sample x 66.66 mg/dl

Antihyperglycemic activity

Male albino rats of the Sprague Dawely Strain (130 g-140 g) were injected intraperitoneally with alloxan (150 mg/kg body weight) to induce diabetes mellitus . Hyperglycemia was assessed after 72 hrs by measuring blood glucose and after 2 and 4 weeks intervals. Animals were divided into 6 groups : f0 irst group: Diabetic rats that served as positive control, second group: Diabetic rats that received 100 mg/ Kg of 100% ethanol of plant extract, third group: Diabetic rats that received 100 mg/Kg of 70% ethanol of plant extract, fourth group: Diabetic rats that received 100 mg/Kg of chloroform of plant extract, fifth group: Diabetic rats that received 100 mg/Kg of ethyl acetate of plant extract, sixth group: Diabetic rats that received 100 mg/Kg of cidophage drug as reference drug. At the end of each study period, blood samples were collected from the retro-orbital venous plexus through the eye canthus of anesthetized rats after an overnight fast. Serum was isolated by centrifugation, and the blood glucose level was measured. [13]

Statistical analysis

The obtained data were analyzed by using the Student's t test. [14]


   Results and Discussion Top


Chromatographic separation of the n-butanol led to the isolation of a new flavonoid glycoside (1) in addition to 5 known flavonoids. The isolated flavonoid compounds showed chromatographic properties and ultraviolet absorption spectra with shift reagents characteristic of flavones.

ESI-MS analysis of compound 1 showed molecular ion peak at m/z 593.181[M+1] + corresponding to C 28 H 32 O 14 . The 1 H- NMR spectrum [Table 1] displayed AA`BB` system of a disubstituted benzene ring at δ 7.98 (d, J=8.45) and 7.12 (d, J=8.45). The spectrum showed also a singlet at δ 6.69 for H-3, a singlet at δ 6.15 assigned for H-6, and a methoxy singlet at δ 3.86. For the glycosyl moiety, the spectrum showed two anomeric protons at δ 4.87 (d, J=7.68) for glucose and at δ 4.66 (d, J=1.5) together with the doublet at δ 1.1 (d, J=5.4) indicated a rhamnosyl group in the molecule. The downfield shift of the anomeric proton of glucose rather than the anomeric proton of rhamnose confirmed that the glucose is the terminal sugar and this was supported by comparison with spectral data of the isomeric compound acacetin 8-C-α-L-rhamnopyranosyl-(1→2)-β-D-glucopyranoside, [15] in which the anomeric proton of rhamnosyl was down field shifted than that of glucose, where the rhamnose is terminal sugar. Further confirmation was achieved by prolonged acid hydrolysis of compound 1 to give glucose in the sugar portion, indicating that the glucose was the terminal sugar. From these data, compound 1 [Figure 1] was identified as acacetin 8-C-β-D-glucopyranosyl-(1→2)-α-L-rhamnopyranoside.
Figure 1: Structure of compound 1

Click here to view
Table 1: ww1H-NMR spectral data of compound 1

Click here to view


The spectral data of the known compounds were in accordance with the published data. They were identified as apigenin 6,8-di-C-β-D-glucopyronoside (vicenin 2), [16] apigenin-7 methyl ether 4′glucoside, [17] apigenin-4`-methyl ether 7-O-rutino-pyranoside, [18] apigenin 7-O-β-D- glucopyranoside, [19] and apigenin. [19]

Bioassay

The 100% ethanol, 70% ethanol, chloroform, and ethyl acetate extracts of A. paniculatum were tested for their different pharmacological activity. Study of the acute toxicity of 70% ethanol extract of A. paniculatum leaves was the most safe up to 5 gm/kg. The anti- inflammatory activity results showed that the ethyl acetate (100 mg/ kg.b.wt.) is the most potent extract as it reduced the edema by 61.3%. These results nearly similar to the reference drug, indomethacin (64.6% change), followed by 100% ethanol (100 mg/kg.b.wt.), ethyl acetate (50 mg/ kg.b.wt.), 70% ethanol (100 mg/ kg.b.wt.), 100%ethanol (50 mg/ kg.b.wt.), 70% ethanol (50 mg/ kg.b.wt.), then chloroform(100 mg/ kg.b.wt.) extracts with percent of change were 59.7%, 54.4%, 54.2%, 47.6%, 41.9%, and 27.8%, respectively. The least effective extract was the chloroform(50 mg/kg.b.wt.), 21.8% [Table 2]. The analgesic activity showed that the percent of change for the reference drug, tramadol, is 59.8%,and the results indicated that the 100% ethanol extract (100 mg/ kg.b.wt.) possess reasonable analgesic activity followed by 70% ethanol (100 mg/ kg.b.wt.), ethyl acetate (100 mg/ kg.b.wt.), 100% ethanol (50 mg/kg.b.wt.), 70% ethanol (50 mg/ kg.b.wt.), ethyl acetate (50 mg/kg.b.wt.) then chloroform (100 mg/ kg.b.wt.) extracts with percent of change were 44.3%, 39.1%, 35.4%, 35.4%, 27.7%, 24.0%, and 18.0%, respectively. The least effective extract was the chloroform (50 mg/kg.b.wt.); the percent of change was 12.4%. Anti-pyretic activity of 100% ethanol extract was the most effective, the percent inhibition was 5.6% after 2 hrs, while it was 6.1% for the reference drug, subsequently by 70% ethanol then ethyl acetate extracts with percent of change 3.1% and 2.6% respectively, while the least anti-pyretic effective extract was the chloroform; the percent of change was [Table 3] 1.6%. Antioxidant activity results showed that the 100% ethanol (100 mg/kg) extract possess highly antioxidant activity; producing a percent of change 3.0% was nearly similar to thereference drug, vitamin E, producing a percent of change of 1.1%, followed by ethyl acetate (100 mg/kg), 100% ethanol (50 mg/kg) and 70% ethanol (100 mg/kg) extract with percent of change were 3.0%, 4.4%, 8.3%, and 10.5%, respectively, followed by the ethyl acetate (50 mg/kg), 70% ethanol (50 mg/ kg), and chloroform (100 mg/kg) with percent change 12.7%, 15.2%, 21.1%, respectively. The least effective extract was the chloroform (50 mg/kg.b.wt.); the percent of change was 27.3% [Table 4]. The anti-hyperglycemic activity of 100% ethanol (100 mg/kg) extract showed a percent change in serum glucose level of 20.4% and 45.1% after 2 and 4 weeks, respectively. The metformin (100 mg/kg) rats group, the reference drug, shows a decrease in serum glucose level by 29.6% and 66.9% after 2 and 4 weeks, respectively. The percent change of 100% ethanol extract after 4 weeks exceeds that the reference drug after 2 weeks. It is evident from the presented results that the 100% ethanol extract possess significantly anti-hyperglycemic activity followed by 70% ethanol (100 mg/kg) then ethyl acetate (100 mg/kg) extract with percent of change 22.3%, 38.0%, and 26.3%, 37.5% after 2 and 4 weeks, respectively. The least effective extract was the chloroform extract with percent of change 13.4% and 24.3% after 2 and 4 weeks.
Table 2: Acute Anti-inflammatory activity of 100% ethanol, 70% ethanol, chloroform and ethyl acetate extracts of A. paniculatum leaves

Click here to view
Table 3: Analgesic effect of 100% ethanol, 70% ethanol, chloroform and ethyl acetate extracts of A. paniculatum leaves

Click here to view
Table 4: Antioxidant activity of 100% ethanol, 70% ethanol, chloroform and ethyl acetate extracts of A. paniculatum leaves in comparison with vitamin E

Click here to view


 
   References Top

1.Gentry AH. Flora Neotropica. Bignoniaceae part I (Tribes Crescentieae and Tourrettieae). Vol. 25. Flora Neotropica Monograph; 1980. p.1-150.  Back to cited text no. 1
    
2.Gentry AH. Flora Neotropica. Bignoniaceae part II (Tribes Tecomeae). Vol. 25. Flora Neotropica Monograph; 1992. p.1- 130.  Back to cited text no. 2
    
3.Lohmann LG. Untangling the phylogeny of neotropical lianas (Bignonieae, Bignoniaceae). Am J Bot 2006;93:304-18.  Back to cited text no. 3
[PUBMED]    
4.Gentry AH. Bignoniaceae. Flora de Veracruz 1982. 24, p.1-222.  Back to cited text no. 4
    
5.Burger W, Gentry A. Family 194. Bignoniaceae. In: Burger W, Editors. Vol. 41. Flora Costaricensis: Fieldiana, Bot., New Series; 2000. p.77-161.  Back to cited text no. 5
    
6.Paget G, Berne's E. Toxicity tests in evaluation of drugs activities cited in the laboratory rat. London: Academic Press; 1964. p. 135-60.  Back to cited text no. 6
    
7.Karber G. Determination of median leathal dose. Arch Exp Pathol Pharmacol 1931;162:480-7.  Back to cited text no. 7
    
8.Winter GA, Risley EA, Nuss GW. Carrageenin-induced edema in hind paw of the rat as an assay for antiiflammatory drugs. Proc Soc Exp Biol Med 1962;111:544-7.  Back to cited text no. 8
    
9.Koster R, Anderson M, Deber J. Method for determination of analgesic activity. Fed Proc 1959;18:412.  Back to cited text no. 9
    
10.Bush IE, Alexander RW. An improved method for the assay of anti-inflammatory substances in rats. Acta Endocrinol (Copenh) 1960;35:268-76.  Back to cited text no. 10
[PUBMED]    
11.Eliasson SG, Samet JM. Alloxan induced neuropathies: Lipid changes in nerve and root fragments. Life Sci 1969;8:493-8.  Back to cited text no. 11
[PUBMED]    
12.Beutler E, Duron O, Kelly BM. Improved method for the determination of blood glutathione. J Lab Clin Med 1963;61:882- 8.  Back to cited text no. 12
[PUBMED]    
13.Trinder P. Estimation of serum glucose and triglycerides by enzymatic method.Am Clin Biochem 1969-6:24.  Back to cited text no. 13
    
14.Spedecor WG, Choehran GW. Statistical methods for the determination of blood glutathione. J Lab Clin Med 1982;61:882-8.  Back to cited text no. 14
    
15.Larionova M, Spengler I, Nogueiras C, Quijano L, Ramiìrez- Gualito K, Corteìs-Guzmaìn F, et al . AC-Glycosylflavon from Piper ossanum, a Compound Conformationally Controlled by CH/π and Other weak Intramolecular Interactions. J Nat Prod 2010;73:1623-7.  Back to cited text no. 15
    
16.Nawwar MA, El-Sissi HI, Barakat HH. Flavonoid constituents of Ephedra alata.Phytochemistry1984;23:2937.  Back to cited text no. 16
    
17.Markus V, Hans G, Franz-CC, and Kenneth RM. Malonylated flavone 5-O-glucosides in the barren sprouts of equisetum arvense. Phytochemistry 1990;29:2555-60.  Back to cited text no. 17
    
18.Chari VM, Jordan M, Wagner H, Thies PW. A 13 C-NMR study of the structure of Acyl-linarin from Valeriana wallichii. Phytochemistry 1977;16:1110.  Back to cited text no. 18
    
19.Mabry TJ, Markham KR, Thomas MB. The Systematic Identification of Flavonoids. Berlin: Springer Verlag: 1970.  Back to cited text no. 19
    


    Figures

  [Figure 1]
 
 
    Tables

  [Table 1], [Table 2], [Table 3], [Table 4]


This article has been cited by
1 Amphipaniculosides AD, triterpenoid glycosides, and amphipaniculoside E, an aliphatic alcohol glycoside from the leaves of Amphilophium paniculatum
Mamdouh Nabil Samy,Hany Ezzat Khalil,Sachiko Sugimoto,Katsuyoshi Matsunami,Hideaki Otsuka,Mohamed Salah Kamel
Phytochemistry. 2015; 115: 261
[Pubmed] | [DOI]
2 the central analgesic and anti-inflammatory activities of the methanolic extract of carthamus oxycantha
bukhari, i.a.
journal of physiology and pharmacology. 2013; 64(3)
[Pubmed]
3 Chemical composition and antioxidant activity of the ethanol extract and purified fractions of cadillo (Pavonia sepioides)
Cristian A. Gasca,Fabio A. Cabezas,Laura Torras,Jaume Bastida,Carles Codina
Free Radicals and Antioxidants. 2013;
[Pubmed] | [DOI]



 

Top
  
 
  Search
 
    Similar in PUBMED
   Search Pubmed for
   Search in Google Scholar for
 Related articles
    Access Statistics
    Email Alert *
    Add to My List *
* Registration required (free)  

 
  In this article
    Abstract
   Introduction
    Materials and Me...
    Results and Disc...
    References
    Article Figures
    Article Tables

 Article Access Statistics
    Viewed3062    
    Printed146    
    Emailed0    
    PDF Downloaded45    
    Comments [Add]    
    Cited by others 3    

Recommend this journal