Chemical components of the Ephedra major from Iran
SUPPLEMENTARY MATERIAL
Chemical components of the Ephedra major from Iran Mahnaz Aghdasia*, Morteza Mofid Bojnoordia, Manijeh Mianabadia and Mohabat Nadafb a
Department of Biology, Faculty of Science, Golestan University, Gorgan, Iran
b
Department of Biology, Payame Noor University (PNU), Tehran, Iran
Abstract Ephedra is a dioecious shrub belongs to the Ephedraceae family of gymnosperms. Almost all commercial applications of Ephedra extracts is derived from the ephedrine alkaloids found in the evergreen stems. The purpose of this study was to compare chemical components (total alkaloid, ephedrine, total phenol, total flavonoid and tannin) of Ephedra major plants during May to October months. The seeds and stems were collected from Bojnoord altitudes in east of Iran. Total alkaloid was separated by solvent and soxhelet extraction method. The results revealed that solvent extraction method is more efficient than soxhelet extraction method. The measurement of chemical components showed significant difference during May to October months. Data from HPLC analysis revealed that while root is depleted of ephedrine, the ephedrine amount in stem organ ranged from 1.50±0.15 to 2.12±0.01 mg/g dry weight. The results indicate that Ephedra major can be as a suitable source of ephedrine. Key words: Ephedra major; stem; alkaloid; ephedrine; Flavonoid
* E.mail: [email protected]
Experimental Plant materials The green stems of Ephedra major were collected during May to October from Bojnoord altitudes (S: 37º 19ʹ 44ʺ and E: 57º 38ʹ 30ʺ) and identified by one of authors, Mohabat Nadaf. A voucher specimen (No.00109) has been deposited at the Environmental Department of Bojnourd Herbarium, Payame Noor University, Bojnourd, IRAN. Then the collected stems were air dried in the shade and powdered. Seeds of Ephedra major were collected from plants growing in their natural condition from Bojnoord-Iran. Then, the seeds were cultured into plastic pots containing garden soil. The pots were incubated at 25±2 ˚C and 16h light/8h dark photoperiod in a greenhouse condition and irrigated every 4 days. Ten seeds were grown in each pot. Preparation of plant extracts Plant extraction was done according to Pourmorad et al., (2006) method. One gram of powdered samples was soaked in 50 ml 80% aqueous methanol for 48 hours at room temperature and the extracts was filtered. The extraction was repeated twice, and the supernatant of tree extracts were combined in a volumetric flask and evaporated under vacuum at 40 ºC using Rotary evaporator pump. Determination of total phenolic content Total phenolic content were measured using the Folin- Ciocalteaumethod (Meda A, 2005). For this purpose, 0.1 ml of extracted solution, 0.1 ml of aqueous Folin-Ciocalteaur reagent (50% v/v) and 2.8 ml distilled water were mixed in a test tube, thoroughly. Then 2 ml 2% sodium carbonate was added. The mixture was kept 30 min at room temperature in the dark before measuring absorbance at 720nm. All measurements were repeated tree times and results were expressed as Gallic acid equivalents. Determination of total Flavonoid content Total flavonoid content was measured by aluminum chloride method as described by Chang et al., (2002). In brief, 0.5 ml of plant extract was mixed with 0.1 ml of 10% aluminum chloride,
0.1 ml of 1M potassium acetate, 1.5 ml of 80% methanol and 2.8 ml of distilled water. The mixture was kept 40 min at room temperature in the dark. The absorbance of the samples was measured at 415 nm. Quercetin was used as the standard flavonoid. All measurements were performed in triplicate and results were expressed as quercetin equivalents. Determination of total tannin content Total Tannin was determined according to Makkar et al., (1993) based on the ability of polypyrrolidone (PVPP) to irreversibly complex tannins. Extractable tannin was assayed as the difference in total phenols (measured by Folin-Ciocateau reagent) before and after treatment with insoluble polyvinyl PVPP. Total tannin was expressed as tannic acid equivalents. Determination of total alkaloid content Solvent extraction: Hundred milligrams of powdered materials were extracted by 10 ml metanol (80%) for 16 hours at room temperature, and the extract was filtered. The extraction was repeated twice, and the supernatant of tree extracts were combined and filtered. The extracts were collected in a volumetric flask and diluted to final volume with 80% methanol. Soxhlet method: The extraction was undertaken with 1 g of powdered plant material and 300 mL of methanol (80%) in a Soxhlet apparatus for 16 h. The extract was concentrated using rotary evaporator under vacuumpump.
Total alkaloid content measurement Total alkaloid content was measured by spectrophotometry method (Shamsa et al., 2008). The extract was dissolved in 5 ml HCL (2N) and then filtered. The filtrate was transferred to a funnel and washed with 10 ml chloroform (3 times). The pH of solution was adjusted to neutral with 0.1 N NaOH. Then 5 ml bromocresol green and 5 ml phosphate buffer was added to this mixture. The mixture was shaken and the complex was extracted with 1, 2, 3, and 4 ml chloroform by vigorous shaking. The extracts were collected in a 10ml volumetric flask and diluted to volume with chloroform. The absorbance of the complex was measured at 470 nm (SHIMADZU UV1800) against the blank.
Ephedrine extraction Ephedrine was extracted from 1 g dry powdered samplesin 10 ml 50% aqueous methanol by shaker at room temperature for 20 minutes. Then extracts were subjected to centrifugation (5 min at 1500 rpm). The extraction was repeated three times. Then, the extracts were combined and diluted to 50ml with 50% methanol. The extracts were filtered using 0.45 m membrane nylon membrane filters (Sigma) before injection to HPLC. HPLC analysis The amount of ephedrine were determined by High performance liquid chromatography (HPLC) (Merck Hitachi) equipped with a Eurospher C18 (250 X 4 mm) column and a UV/VIS detector, a Diod Array pump and a Merck Hitachi7100–L software for peak integration. Mobile phase was a mixture of 0/1% phosphoric acid (pH 4), 25 mM SDS and 40% acetonitrile (10:1 v/v). The mobile phase was filtered using 0.45 m membrane filter and degassed by vacuumed prior to use. The flow rate was 0.8 mL min-1 and ephedrine was detected at 210 nm. All solvents were HPLC grade (Merk). For each analysis, 20 l of samples were injected into HPLC system. The quantity of ephedrine in each sample was determined from the standard curve. Standard solutions Standard solutions were prepared by dissolving 1 mg ephedrine in 10 ml methanol to yield stock solutions. The stock solutions were stored at 4ᵒC before using. Statistical analysis All data were analyzed by using ANOVA. The mean values were compared by the Duncan's multiple range test at ρ=0.05 significant level using SPSS software.
Total alkaloid (mg g -1 DW)
Figures
7 6 5 4 3 2 1 0
Soxhlet extraction Solvent extraction
Month
Figure S1. The effect of extraction method on total alkaloid content of Ephedra major during May to October months. The data are expressed as means ± SD (n=3).
1
0.05
A Ephedrine (mg g-1 DW)
Total alkaloid (mg g-1 DW)
1.2
0.8 0.6 0.4 0.2 0
B 0.04 0.03 0.02 0.01 0
root
stem
root
stem
Figure S2. A) Total alkaloid content and B) ephedrine content in root and stem of 6 weeks old of Ephedra major plant. The data are expressed as means ± SD (n=3).
Total flavonoi- Total alkaloid (mg. g DW)
9
Alkaloid
8 Flavonoid
7 6 5 4 3 2 1 0 May
June
July
August September October
Month Figure S3.Changes pattern of total flavonoid and total alkaloid level during May to October months.
Tabels Table S1.Total phenolic, flavonoid, tannin, ephedrine content and ephedrine/total alkaloid percent of Ephedra major during May to October months (n=3). Month
Total phenol
Total a
flavonoid
Ephedrined
Total b
c
tannin
Ephedrine/total alkaloid ×100
May
44.90±1.31
5.84±0.08
24.5±1.29
2.12±0.01
0.43
June
32.86±0.28
8.40±0.15
15.61±0.21
1.21±0.01
0.27
July
38.54±0.47
7.21±0.10
14.64±0.10
1.83±0.004
0.69
August
36.21±0.14
5.67±0.08
18.50±2.67
1.79±0.09
0.43
September
35.30±0.59
5.30±0.22
10.36±1.30
1.62±0.13
0.28
October
41.13±0.69
4.63±0.41
15.34±0.24
1.50±0.15
0.25
Note: The data are expressed as mean±SD (n=3). aExpressed as milligram of gallic acid per gram dry weight. b
Expressed as milligram of quercetin per gram dry weight. cExpressed as milligram of extract per gram dry weight.
d
Expressed as milligram of ephedrine per gram dry weight.
Table S2.Variance analysis of total alkaloid, total phenol, total flavonoid, total tannin and ephedrine content of Ephedra major during May to October months. Source
Freedom
Alkaloid
Alkaloid
Total
Total
Total
(Soxhlet)
(Solvent)
phenol
flavonoid
tannin
Ephedrine
Month
21
0.058*
4.023*
81.99*
2.001*
90.672*
0.322*
Error
5
0.003
1.224
3.018
0.033
13.214
0.069
Reference: Chang C, Yang M, Wen H, Chern J, 2002. Estimation of total flavonoid content in propolis by two complementary colorimetric methods. J. Food and Drug Analysis. 10: 178- 182. Makkar HPS, Bluemmel M, Borowy NK, Becker K, 1993.Gravimetric determination of tannins and their correlations with chemical and protein precipitation methods. J. Sci. Food Agr. 61, 161–165. Meda A, Lamien CE, Romito M, Millogo J, Nacoulma OG, 2005.Determinaion of the total phenolic, flavonoid and pralin contents in Burkina Fasan honey, as well as their scavenging activity. Food Chem. 91, 571-577. Pourmorad F, Hosseinimehr SJ, Shahabimajd N, 2006. Antioxidant activity, phenol and flavonoid contents of some selected Iranian medicinal plants. African J. Biotech., 5, 1142-1145. Shamsa F, Monsef H, Ghamooshi R,Verdian-rizi M, 2008. Spectrophotometric determination of total alkaloids in some Iranian medicinal plants. Thai. J. Pharm. and Sci. 32, 17-20.
Chemical components of the Ephedra major from Iran Mahnaz Aghdasia*, Morteza Mofid Bojnoordia, Manijeh Mianabadia and Mohabat Nadafb a
Department of Biology, Faculty of Science, Golestan University, Gorgan, Iran
b
Department of Biology, Payame Noor University (PNU), Tehran, Iran
Abstract Ephedra is a dioecious shrub belongs to the Ephedraceae family of gymnosperms. Almost all commercial applications of Ephedra extracts is derived from the ephedrine alkaloids found in the evergreen stems. The purpose of this study was to compare chemical components (total alkaloid, ephedrine, total phenol, total flavonoid and tannin) of Ephedra major plants during May to October months. The seeds and stems were collected from Bojnoord altitudes in east of Iran. Total alkaloid was separated by solvent and soxhelet extraction method. The results revealed that solvent extraction method is more efficient than soxhelet extraction method. The measurement of chemical components showed significant difference during May to October months. Data from HPLC analysis revealed that while root is depleted of ephedrine, the ephedrine amount in stem organ ranged from 1.50±0.15 to 2.12±0.01 mg/g dry weight. The results indicate that Ephedra major can be as a suitable source of ephedrine. Key words: Ephedra major; stem; alkaloid; ephedrine; Flavonoid
* E.mail: [email protected]
Experimental Plant materials The green stems of Ephedra major were collected during May to October from Bojnoord altitudes (S: 37º 19ʹ 44ʺ and E: 57º 38ʹ 30ʺ) and identified by one of authors, Mohabat Nadaf. A voucher specimen (No.00109) has been deposited at the Environmental Department of Bojnourd Herbarium, Payame Noor University, Bojnourd, IRAN. Then the collected stems were air dried in the shade and powdered. Seeds of Ephedra major were collected from plants growing in their natural condition from Bojnoord-Iran. Then, the seeds were cultured into plastic pots containing garden soil. The pots were incubated at 25±2 ˚C and 16h light/8h dark photoperiod in a greenhouse condition and irrigated every 4 days. Ten seeds were grown in each pot. Preparation of plant extracts Plant extraction was done according to Pourmorad et al., (2006) method. One gram of powdered samples was soaked in 50 ml 80% aqueous methanol for 48 hours at room temperature and the extracts was filtered. The extraction was repeated twice, and the supernatant of tree extracts were combined in a volumetric flask and evaporated under vacuum at 40 ºC using Rotary evaporator pump. Determination of total phenolic content Total phenolic content were measured using the Folin- Ciocalteaumethod (Meda A, 2005). For this purpose, 0.1 ml of extracted solution, 0.1 ml of aqueous Folin-Ciocalteaur reagent (50% v/v) and 2.8 ml distilled water were mixed in a test tube, thoroughly. Then 2 ml 2% sodium carbonate was added. The mixture was kept 30 min at room temperature in the dark before measuring absorbance at 720nm. All measurements were repeated tree times and results were expressed as Gallic acid equivalents. Determination of total Flavonoid content Total flavonoid content was measured by aluminum chloride method as described by Chang et al., (2002). In brief, 0.5 ml of plant extract was mixed with 0.1 ml of 10% aluminum chloride,
0.1 ml of 1M potassium acetate, 1.5 ml of 80% methanol and 2.8 ml of distilled water. The mixture was kept 40 min at room temperature in the dark. The absorbance of the samples was measured at 415 nm. Quercetin was used as the standard flavonoid. All measurements were performed in triplicate and results were expressed as quercetin equivalents. Determination of total tannin content Total Tannin was determined according to Makkar et al., (1993) based on the ability of polypyrrolidone (PVPP) to irreversibly complex tannins. Extractable tannin was assayed as the difference in total phenols (measured by Folin-Ciocateau reagent) before and after treatment with insoluble polyvinyl PVPP. Total tannin was expressed as tannic acid equivalents. Determination of total alkaloid content Solvent extraction: Hundred milligrams of powdered materials were extracted by 10 ml metanol (80%) for 16 hours at room temperature, and the extract was filtered. The extraction was repeated twice, and the supernatant of tree extracts were combined and filtered. The extracts were collected in a volumetric flask and diluted to final volume with 80% methanol. Soxhlet method: The extraction was undertaken with 1 g of powdered plant material and 300 mL of methanol (80%) in a Soxhlet apparatus for 16 h. The extract was concentrated using rotary evaporator under vacuumpump.
Total alkaloid content measurement Total alkaloid content was measured by spectrophotometry method (Shamsa et al., 2008). The extract was dissolved in 5 ml HCL (2N) and then filtered. The filtrate was transferred to a funnel and washed with 10 ml chloroform (3 times). The pH of solution was adjusted to neutral with 0.1 N NaOH. Then 5 ml bromocresol green and 5 ml phosphate buffer was added to this mixture. The mixture was shaken and the complex was extracted with 1, 2, 3, and 4 ml chloroform by vigorous shaking. The extracts were collected in a 10ml volumetric flask and diluted to volume with chloroform. The absorbance of the complex was measured at 470 nm (SHIMADZU UV1800) against the blank.
Ephedrine extraction Ephedrine was extracted from 1 g dry powdered samplesin 10 ml 50% aqueous methanol by shaker at room temperature for 20 minutes. Then extracts were subjected to centrifugation (5 min at 1500 rpm). The extraction was repeated three times. Then, the extracts were combined and diluted to 50ml with 50% methanol. The extracts were filtered using 0.45 m membrane nylon membrane filters (Sigma) before injection to HPLC. HPLC analysis The amount of ephedrine were determined by High performance liquid chromatography (HPLC) (Merck Hitachi) equipped with a Eurospher C18 (250 X 4 mm) column and a UV/VIS detector, a Diod Array pump and a Merck Hitachi7100–L software for peak integration. Mobile phase was a mixture of 0/1% phosphoric acid (pH 4), 25 mM SDS and 40% acetonitrile (10:1 v/v). The mobile phase was filtered using 0.45 m membrane filter and degassed by vacuumed prior to use. The flow rate was 0.8 mL min-1 and ephedrine was detected at 210 nm. All solvents were HPLC grade (Merk). For each analysis, 20 l of samples were injected into HPLC system. The quantity of ephedrine in each sample was determined from the standard curve. Standard solutions Standard solutions were prepared by dissolving 1 mg ephedrine in 10 ml methanol to yield stock solutions. The stock solutions were stored at 4ᵒC before using. Statistical analysis All data were analyzed by using ANOVA. The mean values were compared by the Duncan's multiple range test at ρ=0.05 significant level using SPSS software.
Total alkaloid (mg g -1 DW)
Figures
7 6 5 4 3 2 1 0
Soxhlet extraction Solvent extraction
Month
Figure S1. The effect of extraction method on total alkaloid content of Ephedra major during May to October months. The data are expressed as means ± SD (n=3).
1
0.05
A Ephedrine (mg g-1 DW)
Total alkaloid (mg g-1 DW)
1.2
0.8 0.6 0.4 0.2 0
B 0.04 0.03 0.02 0.01 0
root
stem
root
stem
Figure S2. A) Total alkaloid content and B) ephedrine content in root and stem of 6 weeks old of Ephedra major plant. The data are expressed as means ± SD (n=3).
Total flavonoi- Total alkaloid (mg. g DW)
9
Alkaloid
8 Flavonoid
7 6 5 4 3 2 1 0 May
June
July
August September October
Month Figure S3.Changes pattern of total flavonoid and total alkaloid level during May to October months.
Tabels Table S1.Total phenolic, flavonoid, tannin, ephedrine content and ephedrine/total alkaloid percent of Ephedra major during May to October months (n=3). Month
Total phenol
Total a
flavonoid
Ephedrined
Total b
c
tannin
Ephedrine/total alkaloid ×100
May
44.90±1.31
5.84±0.08
24.5±1.29
2.12±0.01
0.43
June
32.86±0.28
8.40±0.15
15.61±0.21
1.21±0.01
0.27
July
38.54±0.47
7.21±0.10
14.64±0.10
1.83±0.004
0.69
August
36.21±0.14
5.67±0.08
18.50±2.67
1.79±0.09
0.43
September
35.30±0.59
5.30±0.22
10.36±1.30
1.62±0.13
0.28
October
41.13±0.69
4.63±0.41
15.34±0.24
1.50±0.15
0.25
Note: The data are expressed as mean±SD (n=3). aExpressed as milligram of gallic acid per gram dry weight. b
Expressed as milligram of quercetin per gram dry weight. cExpressed as milligram of extract per gram dry weight.
d
Expressed as milligram of ephedrine per gram dry weight.
Table S2.Variance analysis of total alkaloid, total phenol, total flavonoid, total tannin and ephedrine content of Ephedra major during May to October months. Source
Freedom
Alkaloid
Alkaloid
Total
Total
Total
(Soxhlet)
(Solvent)
phenol
flavonoid
tannin
Ephedrine
Month
21
0.058*
4.023*
81.99*
2.001*
90.672*
0.322*
Error
5
0.003
1.224
3.018
0.033
13.214
0.069
Reference: Chang C, Yang M, Wen H, Chern J, 2002. Estimation of total flavonoid content in propolis by two complementary colorimetric methods. J. Food and Drug Analysis. 10: 178- 182. Makkar HPS, Bluemmel M, Borowy NK, Becker K, 1993.Gravimetric determination of tannins and their correlations with chemical and protein precipitation methods. J. Sci. Food Agr. 61, 161–165. Meda A, Lamien CE, Romito M, Millogo J, Nacoulma OG, 2005.Determinaion of the total phenolic, flavonoid and pralin contents in Burkina Fasan honey, as well as their scavenging activity. Food Chem. 91, 571-577. Pourmorad F, Hosseinimehr SJ, Shahabimajd N, 2006. Antioxidant activity, phenol and flavonoid contents of some selected Iranian medicinal plants. African J. Biotech., 5, 1142-1145. Shamsa F, Monsef H, Ghamooshi R,Verdian-rizi M, 2008. Spectrophotometric determination of total alkaloids in some Iranian medicinal plants. Thai. J. Pharm. and Sci. 32, 17-20.
