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Flavonoids of four Cleome and three Capparis species
BiochemicalSystematicsandEcology,Vol. 25, No. 2, pp. 161-166, 1997 © 1997 ElsevierScience Ltd All rights reserved.Printed in Great Britain 0305-1978/97 $17.00+0.00
Pergamon PII: S0305-1978(96)00099-3
Flavonoids of Four Cleome and Three Capparis Species MOHAMED SHARAF, MOHAMED A. EL-ANSARI and NABIEL A. M. SALEH* Department of Plant Systematic and Egyptian Flora, National Research Centre, EI-Dokki, Cairo, Egypt
Key Word Index--Cleome; Capparis; Cleomaceae; Capparaceae; flavonoids. A b s t r a c t - - T h e aerial parts of four Cleome and three Capparis species were investigated for their flavonoid constituents. Thirteen flavonoid glycosides were isolated and identified as kaempferol 7-rhamnoside, kaempferol 3-rutinoside, kaempferol 3,7-dirhamnoside, kaempferol 3-glucoside-7-rhamnoside, kaempferol 3-rhamnoside-7-glucoside, quercetin 7-rhamnoside, quercetin 3-rutinoside, quercetin 7-rutinoside, quercetin 3,7dirhamnoside, quercetin 3-glucoside-7-rhamnoside, isorhamnetin 3-rutinoside, isorhamnetin 3,7-dirhamnoside, and apigenin 6,8-di-C-glucoside. © 1997 Elsevier Science Ltd
Introduction According to Mabberley (1990) and Willis (1966), Cleome (L.) DC. is a large genus with 1 50 species in tropical countries in both the Old and New Worlds. However, the familial assignment of the genus is rather debatable. Although Cleome and 11 other genera (the chief of which are Cleomella, Gynandropsis and Physostemon) have been treated as a separate family Cleomaceae (Pax) Airy Shaw (Taeckholm, 1 974), Cleome is usually included in the Capparaceae (Greuter et al., 1989). This is based on the fact that Cleomaceae differs from most Capparaceae in the glandular covering and in the fruit being "'cruciferous'" siliqua with a replum, and from the Brassicaceae, tribe Stanleyeae, in the digitately three- to seven-foliolate, rarely simple leaves, often zygomorphic flowers, and rarely tetradynamous stamens. Cleome amplyocarpa is commonly known as the spider flower, and in Arabic as magnuna or wahhsh (Boulos, 1983). Its leaf infusion is reported to have an almost immediate effect on relieving abdominal and rheumatic pains (Boulos, 1983). In a previous study, we reported the isolation and identification of the methylated flavonoids from four Cleome species (Sharaf et al., 1992). In the present communication the structure elucidation of the flavonoid glycosides from three Capparis and the same four Cleome species is reported. M a t e r i a l s and M e t h o d s Plant material. Fresh plant material was collected from the following localities: Cleome droserifolia (Forssk.) Del., Wadi EI-Gaib along the Dahab-Saint Catherine road in June; C. amplyocarpa Barr. and Murb., southern Sinai in April; C. brachycarpa Vahl., EI-Taaif, Saudi Arabia in August; C. chrysantha Decne., along DahabSharm-El-Sheikh road in June; Capparis cam'logenia Decne, along Dahab-Sharm-EI-Sheikh road in June; C. decidua, Wadi Hodein in April; and C. spinosa, eastern Sinai, mountain slopes in August. Voucher specimens are deposited at the herbarium of the NRC as well as that of the University of Cairo. Isolation and identification of flavonoids. Dried leaves and stems were extracted with 70% EtOH Concentrated extracts were fractionated using polyamide column chromatography followed by preparative elution on filter paper (3MM). Purification was carried out on Sephadex LH-20. Solvents used for paper chromato*Corresponding author.
(Received 23 April 1 996; accepted 30 September 1996) 161
162
M. SHARAFETAL.
graphy were: (1) n-butanol:acetic acid:water (BAW, 4:1:5), (2) acetic acid:water (17:3), (3) phenol:water (4:1), (4) chloroform:acetic acid:water (CAW, 13:6:1 ). Flavonoids were detected by their absorbance under UV light. Standard methods of identification were followed (Harborne, 1967; Mabry et al., 1970; Markham, 1982). 1H-NMR spectra were recorded in DMSO-d8 on a Bruker 270 MHz instrument. Mass spectra were recorded using a Finnigan SSQ-7000 (El and CI modes) and Kratos MS 80 (FAB mode).
Results and Discussion Preliminary studies showed that the species under investigation contained no flavanones (negative sodium/borohydride-HCI test) and no sulfated flavonoids (no mobility on electrophoresis). Furthermore, the three Capparis species under investigation contained none of the methylated flavonoids previously isolated from the Cleome species (1-1 O, see Table 1 ) (Sharaf et al., 1992). Thirteen flavonoid glycosides (11-23) were isolated and identified (Table 1 ). Compounds 11-15 on acid hydrolysis gave kaempferol as the aglycone. The sugar moiety was rhamnose for 11 and 13, and additional glucose for 12, 14, and 15. The 1H-NMR spectra of compounds 11-15 showed the characteristic aglycone pattern of kaempferol derivatives (Table 3). For example, the 1H-NMR spectrum of compound 14 showed a two-proton doublet for H-2' and H-6' at 8 8.10 (d, J = 9 Hz) coupled to another doublet at 56.90 (d, J = 9 Hz) for H-3' and H-5', whereas H-6 and H-8 appeared at 56.45 and 6.77 (d, J = 2 Hz). The anomeric protons of glucose and rhamnose appeared at 55.29 (d, J = 7 Hz) and 55.50 (s). The three protons" signal that appeared as a doublet (d, J = 6 H z ) at 50.90 was assigned to the rhamnose methyl. CI-MS of 14 showed a positive molecular ion peak [M + 1]+ at m/e = 595 (35%), and fragments at m / e = 431 (34%) after the loss of a glucose unit, and at m/e = 287 for the aglycone after the loss of the rhamnose moiety. On the basis of the chemical, UV, MS and 1H-NMR studies, compounds 11-15 were identified as kaempferol 7-rhamnoside, kaempferol 3-rutinoside, kaempferol 3,7-dirhamnoside, kaempferol 3-glucoside-7-rhamnoside, and kaempferol 3-rhamnoside-7-glucoside, respectively. Acid hydrolysis of compounds 16 and 19 gave quercetin, rhamnose, and additional glucose for 17, 18 and 20, all of which co-chromatographed with an authentic sample. The results of chemical studies on the isolated compounds are shown in Table 2. UV spectral studies with diagnostic shift reagents (Mabry et al., 1970) indicated that the glycosylation is at position 7 for compounds 16 and 18, position 3 for 17, and at position 3,7 for compounds 19 and 20. Thus, compounds 16 and 19 were identified as quercetin 7-rhamnoside and quercetin 3,7-dirhamnoside, respectively. H 2 0 2 oxidation of 17 and 18 gave a disaccharide, which, from its Rf value and cochromatography with an authentic sample, was proved to be rutinose, Thus compounds 17 and 18 were identified as quercetin 3-rutinoside and quercetin 7-rutinoside, respectively. Enzymatic hydrolysis with J3-glucosidase of 20 (Table 2) gave an intermediate, which was identified as quercetin 7-rhamnoside. H 2 0 2 oxidation for 3h afforded glucose. Thus compound 20 is identified as quercetin 3-glucoside-7-rhamnoside. The 1H-NMR and mass spectra are in a good agreement with the proposed structures (Tables 3 and 4). The 1H-NMR spectrum of compounds 21 and 22 indicated that the flavonoid nucleus is isorhamnetin. This was confirmed by co-chromatography of the aglycone released after acid hydrolysis with an authentic sample. The sugar moiety was rhamnose and glucose for 21, and rhamnose for 22. ~H-NMR spectra indicated that both have a substitution at position 3 (see Table 3). The UV spectra of 21 and 22 (Mabry et al., 1970)
+ tr tr + + + + + + +
3. 5 , 7 , 4 ' - T r i h y d r o x y - 6 , 3 ' - d i m e t h o x y f l a v o n e
4. 5 , 4 ' - D i h y d r o x y - 3 , 6 , 7 - t r i m e t h o x y f l a v o n e
5. 5 , 7 , 3 ' , 4 ' - T e t r a h y d r o x y - 3 , 6 - d l m e t h o x y f lavo n e
6.5,7,4'-Trihydroxy-6,3',5'-trimethoxyflavone
7.5,4'-Dihydroxy-3,6,7,3'-tetramethoxyflavone
8. 5 , 3 ' - D i h y d r o x y - 3 , 6 , 7 , 4 ' , 5 ' - p e n t a m e t h o x y f l a v o n e
tr
++ + +
16. Kaempferol 3-Rha-7-G
16. Quercetin 7-Rha
19. Quercetin 3,7-diRha
+ +
23. Apigenin 6,8-di-C-G
+ + + = M a j o r ; + + = s t r o n g ; + = p r e s e n t ; tr = trace; -
= absent.
+
22. Isorhamnetin 3,7-diRha
G = Glucoside; Rha = rhamnoside; Rut = rutinoside.
+ +
21. Isorhamnetin 3-Rut +
+ +
+ +
-
-
18. Quercetin 7-Rut
20. Quercetin 3-G-7-Rha
++ ++
17. Quercetin 3-Rut + +
+
14. Kaempferol 3-G-7-Rha
+ ++
++
_
+ +
_
_
+++
+
+
+
_
tr +++
+ + +
+ + +
+ + +
+ + +
+ +
+ +
+++
C. droserifolia
+
_
+ +
_
_
++
+
+
++
+
_
++
+ +
13. Kaempferol 3,7-diRha
+
tr +
_
_
+ + +
_
tr
tr
tr
C. chrysantha
_
+
11. Kaempferol 7-Rha
tr
+ +
_
+ + +
-
tr
tr
+
C. brachycarpa
12. Keempferol 3-Rut
+ +
10. 5-Hydroxy-3,6,7,3',4',5'-hexamethoxyflavona
9. 5 , 4 ' - D i h y d r o x y - 3 , 6 , 7 , 8 , 3 ' - p e n t a m e t h o x y f l a v o n e
species
C. amplyocarpa
Cleome
CLEOMEAND CAPPARIS S P E C I E S
2. 5 , 7 , 4 ' - T r i h y d r o x y - 3 , 3 ' - d i m e t h o x y f l a v o n e
IN
tr
OF THE FLAVONOIDS
1.5,7,4'-Trihydroxy-3-methoxyflavone
Compound
T A B L E 1. D I S T R I B U T I O N
-
-
+ + +
-
+ +
+ +
-
-
-
-
+ + +
-
-
-
-
-
-
-
-
C. a/lilogenia
Capparis s p e c i e s
_
_
+
_
+ +
+
_
_
_
_
+
_
_
_
_
_
_
_
_
C. decidua
_
_
+
_
+
+++
_
_
_
_
÷+
_
_
_
_
_
_
_
_
C. spinosa
-I .I~o
~> z
o -11
¢n
< O z O
kaempferol, rhamnose kaempferol, glucose, rhamnose kaempferol, rhamnose kaempferol, glucose, rhamnose kaempferol, glucose, rhamnose quercetin, rhamnose quercetin, glucose, rhamnose quercetin, glucose, rhamnose quercetin, rhamnose quercetin, glucose, rhamnose isorhamnetin, glucose, rhamnose isorhamnetin, rhamnose unchanged
11 Kaempferol 7-Rha 12. Kaempferol 3-Rut 13. Kaempferol 3,7-diRha 14 Kaempferol 3-G-7-Rha 15. Kaempferol 3-Rha-7-G 16. Quercetin 7-Rha 17, Quercetin 3-Rut 18. Quercetin 7-Rut 19. Quercetin 3,7-diRha 20 Quercetin 3-G-7-Rha 21, Isorhamnetin 3-Rut 22. Isorhamnetin 3,7-diRha 23. Apigenin 6,8-di-C-G
G = Glucoside; Rha = rhamnoside; Rut = rutinoside
Complete acid hydrolysis
Compound kaempferol kaempferol, kaempferol 3-glucoside kaempferol 7-rhamnoside kaempferol, kaempferol 7-rhamnoside kaempferol, kaempferol 7-glucoside quercetin quercetin, quercetin 3-glucoside quercetin, quercetin 7-glucoside quercetin 7-rhamnoside quercetin, quereetin 7-rhamnoside isorhamnetin, isorhamnetin 3-glucoside isorhamnetin, isorhamnetin 7-rhamnoside unchanged
Mild acid hydrolysis
TABLE 2, CHEMICAL STUDIES OF THE ISOLATED FLAVONOID GLYCOSIDES
negative negative negative kaempfero! 7-rhamnoside kaempferol 3-rhamnoside negative negative negative negative quercetin 7-rhamnoside negative negative unchanged
Enzymatic hydrolysis
-rutinose rhamnose glucose glucose -rutinose rutinose rhamnose glucose rhamnose rhamnose unchanged
Hydrogen peroxide oxidation
----
-----
----
6.75 (s)--
13 14 15
16 17 18 19
20 21 22
23
(d, J= 2Hz) (d, J= 2Hz) (d, J= 2Hz) (s)
"See Table 1 for structures.
6.20(d,J=2Hz) 6.20(d,J=2Hz) 6.45 (d, J= 2 Hz)
6.20 5.20 6.30 6.45
6.45(d,J=2Hz) 5,45(d,J=2Hz) 5.48 (d, J= 2Hz)
6.40(d,J=2Hz) 6,20 (d, J=2Hz)
---
11 12
H-6
H-3
Compound no."
(d, J = 2 Hz) (d, J = 2 Hz) (d, J=2Hz) (s)
--
6.40(d,J=2Hz) 6.40(d,J=2Hz) 6,75 (d, J=2Hz)
6.42 6.40 5.45 6.75
6.82(d,J=2Hz) 6.77(d,J=2Hz) 6.77 (d, J = 2 Hz)
6.82(d,J=2Hz) 6.35 (d, J = 2 Hz)
H-8
(d, J=gHz) (m) (m) (m)
8.00 (m)
7.45(m) 7,80(s) 7.45 (m)
7.65 7.50 7.40 7.35
7.80(d.J=9Hz) 8.10(d,J=gHz) 8.10 (d, J=9Hz)
8.10(d,J=9Hz) 8.00 (d, J=9Hz)
H-2'
TABLE 3~ 1 H - N M R DATA OF THE ISOLATED FLAVONOID GLYCOSIDES
6.90 (d, J= 9 Hz)
----
-----
6.95(d,J=gHz) 6.90(d,J=9Hz) 6.90 (d, J = 9 Hz)
6.95(d,J=9Hz) 6.85 (d, J=9Hz)
H-3'
(d, J=9Hz) (d, J= 9 Hz) (d, J= 9 Hz) (d, J = 9 Hz)
6.90 (d, J = 9 Hz)
5.82(dd, J=9Hz) 6.90(dd, J=9Hz) 6.95 (d, J = 9 Hz)
6.90 6.82 5.80 6.93
6.95(d,J=gHz) 6.90(d,J=9Hz) 5,90 (d, J=9Hz)
6.95(d,J=9Hz) 6.85 (d, J= 9 Hz)
H-5'
8.00 (m)
7.45(m) 7.50(d,J=9Hz) 7.45 (m)
7.65 dd 7.50 (m) 7.40 (m) 7.35 (m)
7.80(d,J=gHz) 8.10(d,J=9Hz) 8.10 (d, J = 9 Hz)
8.10(d,J=9Hz) 8.00 (d, J= 9 Hz)
H-6'
5.53(s) 4.30 (d,J=2 Hz)
H-1 r h a m n o s e
1.1 (d,J=6Hz) 0.98 (d, J= 6 Hz)
CH3-rhamnose
4.20 4.20 4.50 5.60
(d, J = 2 Hz) (d, J=2Hz) (d, J= 2.5 Hz) (s), 5.40 (s)
0.90 (d, J = 6 Hz) 1.00 (d, J = 6 Hz) 0.95 (d, J = 6 Hz) 1,30, 0.95 (d, J= 6 Hz)
4,55, 5,20
--
--
5.15(d,J=7.5Hz) 4.40(s) 1.00(d,J=6Hz) 5.40(d,J=7Hz) 4.45(s) 0.99(d,J=SHz) -5.58, 5.40 (d, J= 2 Hz) 1.30, 0.95 (d, J= 6 Hz)
-5.15 (d, J = 7 Hz) 5.2 (d,J=7 Hz) --
-5.55,5.30(d,J=2Hz) 1.14,0.82(d,J=6Hz) 5.29(d,J=7.5Hz) 5.50(d,J=2Hz) 0.90(d,J=6Hz) 5.29 (d, J=7.5 Hz) 5.50 (d, J = 2 Hz) 0.90 (d, J= 6 Hz)
-5.30 (d, J = 7 Hz)
H-1 glucose
M. SHARAFETAL.
166
TABLE 4. MS DATA OF THE ISOLATED FLAVONOIDS GLYCOSIDES Compound no."
11
12
13
14
15
19
[M +1]+ [M+1-292]* M+ [M-308] [M + 1 - 162] ÷ [M + 1 - 308] + [ M - 162] + [M + 1 - 146] + [M+1-182] + [M+ 1-36] +
433 (47)
595 (18)
579 (22) 287 (46)
595 (3)
595 (35)
595 (1) 303 (35)
594 (100)
20
22
317 (27) 610 (55) 302 (2)
433 (34) 287 (3)
287 (100)
448 (13) 287 (46)
433 (47) 413 (8) 573 (7)
*See Table 1 for structures. FAB-mode was carried out for compounds 11 and 13, positive CI-mode for compounds 12, 14, 15, 19 and 22, while El-mode was carried out for compound 20.
indicated that compound 21 has a substitution at position 3, and at 3,7 positions for 22. Thus compound 22 is identified as isorhamnetin 3,7-dirhamnoside. H202 oxidation for 3 h of compound 21 afforded the disaccharide rutinose (Rt value, co-chromatography). Thus compound 21 is identified as isorhamnetin 3-rutinoside. 1H-NMR and MS supported the proposed structures (Tables 3 and 4). Finally, compound 23 is identified as apigenin 6,8-di-C-glucoside from its 1H-NMR and co-chromatography with an authentic sample. In conclusion, the chemistry of the four Cleome species and the three Capparis species investigated showed a clear difference in their flavonoid profile. Cleome species showed the presence of 10 methylated flavonoids and eight flavonol glycosides. On the other hand, only four flavonol glycosides were detected in Capparis species. Little has been reported on the flavonoids of members of the Capparaceae. Capparis spinosa is reported to contain rutin (quercetin 3-rutinoside) (Ahmed et al., 1972) as well as quercetin 7rutinoside (Artemeva et al., 1981). Both flavonoids were found to be present in the current study along with kaempferol 3-rutinoside and quercetin 3-glucoside-7-rhamnoside in C. spinosa. The present results appear to support Taeckholm's (1974) classification of Cleome under the family Cleomaceae. References Ahmed, Z. F., Rizk, A. M., Hammouda, F. M. and Seif-EI-Nasr, M. M. (1972) Glucosinolates of Egyptian Capparis species.Phytochemistry 11,251-256. Artemeva, M. V., Karryev, M. O., Meshcheryakov,A. A. and Gordienko,V. P. (1981 ) A new flavonol glycoside, quercetin 7-O-glucorhamnosidefrom Capparisspinosa. Izv. Akad. Nauk Turkm. SSSR, Set. Fizl- Tekh. 3, 123. Boulos, L. (1983) Medicinal Plants of North Africa, p. 52. ReferencePublications Inc., Algonac, Michigan. Greuter, W., Burdet, H. M. and Long, C. (eds) (1989) Med-Checklist. Conservatoireet Jardin botaniques, Geneva. Harborne, J. B. (1967) ComparativeBiochemistry of the Flavonoids. Academic Press, London. Mabberley, D. J. (1990) The PlantBook. Cambridge University Press,Cambridge. Mabry, T. J., Markham, K. R. and Thomas, M. B. (1970). The Systematic Identification of Flavonoids. Springer, Berlin. Markham, J. R. (1982) Techniquesof Flavonoid Identification. Academic Press,London. Sharaf, M., Mansour, R. M. A. and Saleh, N. A. M. (1992) Exudateflavonoids from aerial parts of four Cleome species. Biochem. Syst. Ecol. 20, 443-448. Taeckholm, V. (1974) Student Flora of Egypt, 2nd edn, pp. 167-169. CooperativePrinting Company, Beirut. Willis, J. C. (1966) A Dictionary of the Flowering Plants and Ferns. Cambridge University Press, Cambridge.
Pergamon PII: S0305-1978(96)00099-3
Flavonoids of Four Cleome and Three Capparis Species MOHAMED SHARAF, MOHAMED A. EL-ANSARI and NABIEL A. M. SALEH* Department of Plant Systematic and Egyptian Flora, National Research Centre, EI-Dokki, Cairo, Egypt
Key Word Index--Cleome; Capparis; Cleomaceae; Capparaceae; flavonoids. A b s t r a c t - - T h e aerial parts of four Cleome and three Capparis species were investigated for their flavonoid constituents. Thirteen flavonoid glycosides were isolated and identified as kaempferol 7-rhamnoside, kaempferol 3-rutinoside, kaempferol 3,7-dirhamnoside, kaempferol 3-glucoside-7-rhamnoside, kaempferol 3-rhamnoside-7-glucoside, quercetin 7-rhamnoside, quercetin 3-rutinoside, quercetin 7-rutinoside, quercetin 3,7dirhamnoside, quercetin 3-glucoside-7-rhamnoside, isorhamnetin 3-rutinoside, isorhamnetin 3,7-dirhamnoside, and apigenin 6,8-di-C-glucoside. © 1997 Elsevier Science Ltd
Introduction According to Mabberley (1990) and Willis (1966), Cleome (L.) DC. is a large genus with 1 50 species in tropical countries in both the Old and New Worlds. However, the familial assignment of the genus is rather debatable. Although Cleome and 11 other genera (the chief of which are Cleomella, Gynandropsis and Physostemon) have been treated as a separate family Cleomaceae (Pax) Airy Shaw (Taeckholm, 1 974), Cleome is usually included in the Capparaceae (Greuter et al., 1989). This is based on the fact that Cleomaceae differs from most Capparaceae in the glandular covering and in the fruit being "'cruciferous'" siliqua with a replum, and from the Brassicaceae, tribe Stanleyeae, in the digitately three- to seven-foliolate, rarely simple leaves, often zygomorphic flowers, and rarely tetradynamous stamens. Cleome amplyocarpa is commonly known as the spider flower, and in Arabic as magnuna or wahhsh (Boulos, 1983). Its leaf infusion is reported to have an almost immediate effect on relieving abdominal and rheumatic pains (Boulos, 1983). In a previous study, we reported the isolation and identification of the methylated flavonoids from four Cleome species (Sharaf et al., 1992). In the present communication the structure elucidation of the flavonoid glycosides from three Capparis and the same four Cleome species is reported. M a t e r i a l s and M e t h o d s Plant material. Fresh plant material was collected from the following localities: Cleome droserifolia (Forssk.) Del., Wadi EI-Gaib along the Dahab-Saint Catherine road in June; C. amplyocarpa Barr. and Murb., southern Sinai in April; C. brachycarpa Vahl., EI-Taaif, Saudi Arabia in August; C. chrysantha Decne., along DahabSharm-El-Sheikh road in June; Capparis cam'logenia Decne, along Dahab-Sharm-EI-Sheikh road in June; C. decidua, Wadi Hodein in April; and C. spinosa, eastern Sinai, mountain slopes in August. Voucher specimens are deposited at the herbarium of the NRC as well as that of the University of Cairo. Isolation and identification of flavonoids. Dried leaves and stems were extracted with 70% EtOH Concentrated extracts were fractionated using polyamide column chromatography followed by preparative elution on filter paper (3MM). Purification was carried out on Sephadex LH-20. Solvents used for paper chromato*Corresponding author.
(Received 23 April 1 996; accepted 30 September 1996) 161
162
M. SHARAFETAL.
graphy were: (1) n-butanol:acetic acid:water (BAW, 4:1:5), (2) acetic acid:water (17:3), (3) phenol:water (4:1), (4) chloroform:acetic acid:water (CAW, 13:6:1 ). Flavonoids were detected by their absorbance under UV light. Standard methods of identification were followed (Harborne, 1967; Mabry et al., 1970; Markham, 1982). 1H-NMR spectra were recorded in DMSO-d8 on a Bruker 270 MHz instrument. Mass spectra were recorded using a Finnigan SSQ-7000 (El and CI modes) and Kratos MS 80 (FAB mode).
Results and Discussion Preliminary studies showed that the species under investigation contained no flavanones (negative sodium/borohydride-HCI test) and no sulfated flavonoids (no mobility on electrophoresis). Furthermore, the three Capparis species under investigation contained none of the methylated flavonoids previously isolated from the Cleome species (1-1 O, see Table 1 ) (Sharaf et al., 1992). Thirteen flavonoid glycosides (11-23) were isolated and identified (Table 1 ). Compounds 11-15 on acid hydrolysis gave kaempferol as the aglycone. The sugar moiety was rhamnose for 11 and 13, and additional glucose for 12, 14, and 15. The 1H-NMR spectra of compounds 11-15 showed the characteristic aglycone pattern of kaempferol derivatives (Table 3). For example, the 1H-NMR spectrum of compound 14 showed a two-proton doublet for H-2' and H-6' at 8 8.10 (d, J = 9 Hz) coupled to another doublet at 56.90 (d, J = 9 Hz) for H-3' and H-5', whereas H-6 and H-8 appeared at 56.45 and 6.77 (d, J = 2 Hz). The anomeric protons of glucose and rhamnose appeared at 55.29 (d, J = 7 Hz) and 55.50 (s). The three protons" signal that appeared as a doublet (d, J = 6 H z ) at 50.90 was assigned to the rhamnose methyl. CI-MS of 14 showed a positive molecular ion peak [M + 1]+ at m/e = 595 (35%), and fragments at m / e = 431 (34%) after the loss of a glucose unit, and at m/e = 287 for the aglycone after the loss of the rhamnose moiety. On the basis of the chemical, UV, MS and 1H-NMR studies, compounds 11-15 were identified as kaempferol 7-rhamnoside, kaempferol 3-rutinoside, kaempferol 3,7-dirhamnoside, kaempferol 3-glucoside-7-rhamnoside, and kaempferol 3-rhamnoside-7-glucoside, respectively. Acid hydrolysis of compounds 16 and 19 gave quercetin, rhamnose, and additional glucose for 17, 18 and 20, all of which co-chromatographed with an authentic sample. The results of chemical studies on the isolated compounds are shown in Table 2. UV spectral studies with diagnostic shift reagents (Mabry et al., 1970) indicated that the glycosylation is at position 7 for compounds 16 and 18, position 3 for 17, and at position 3,7 for compounds 19 and 20. Thus, compounds 16 and 19 were identified as quercetin 7-rhamnoside and quercetin 3,7-dirhamnoside, respectively. H 2 0 2 oxidation of 17 and 18 gave a disaccharide, which, from its Rf value and cochromatography with an authentic sample, was proved to be rutinose, Thus compounds 17 and 18 were identified as quercetin 3-rutinoside and quercetin 7-rutinoside, respectively. Enzymatic hydrolysis with J3-glucosidase of 20 (Table 2) gave an intermediate, which was identified as quercetin 7-rhamnoside. H 2 0 2 oxidation for 3h afforded glucose. Thus compound 20 is identified as quercetin 3-glucoside-7-rhamnoside. The 1H-NMR and mass spectra are in a good agreement with the proposed structures (Tables 3 and 4). The 1H-NMR spectrum of compounds 21 and 22 indicated that the flavonoid nucleus is isorhamnetin. This was confirmed by co-chromatography of the aglycone released after acid hydrolysis with an authentic sample. The sugar moiety was rhamnose and glucose for 21, and rhamnose for 22. ~H-NMR spectra indicated that both have a substitution at position 3 (see Table 3). The UV spectra of 21 and 22 (Mabry et al., 1970)
+ tr tr + + + + + + +
3. 5 , 7 , 4 ' - T r i h y d r o x y - 6 , 3 ' - d i m e t h o x y f l a v o n e
4. 5 , 4 ' - D i h y d r o x y - 3 , 6 , 7 - t r i m e t h o x y f l a v o n e
5. 5 , 7 , 3 ' , 4 ' - T e t r a h y d r o x y - 3 , 6 - d l m e t h o x y f lavo n e
6.5,7,4'-Trihydroxy-6,3',5'-trimethoxyflavone
7.5,4'-Dihydroxy-3,6,7,3'-tetramethoxyflavone
8. 5 , 3 ' - D i h y d r o x y - 3 , 6 , 7 , 4 ' , 5 ' - p e n t a m e t h o x y f l a v o n e
tr
++ + +
16. Kaempferol 3-Rha-7-G
16. Quercetin 7-Rha
19. Quercetin 3,7-diRha
+ +
23. Apigenin 6,8-di-C-G
+ + + = M a j o r ; + + = s t r o n g ; + = p r e s e n t ; tr = trace; -
= absent.
+
22. Isorhamnetin 3,7-diRha
G = Glucoside; Rha = rhamnoside; Rut = rutinoside.
+ +
21. Isorhamnetin 3-Rut +
+ +
+ +
-
-
18. Quercetin 7-Rut
20. Quercetin 3-G-7-Rha
++ ++
17. Quercetin 3-Rut + +
+
14. Kaempferol 3-G-7-Rha
+ ++
++
_
+ +
_
_
+++
+
+
+
_
tr +++
+ + +
+ + +
+ + +
+ + +
+ +
+ +
+++
C. droserifolia
+
_
+ +
_
_
++
+
+
++
+
_
++
+ +
13. Kaempferol 3,7-diRha
+
tr +
_
_
+ + +
_
tr
tr
tr
C. chrysantha
_
+
11. Kaempferol 7-Rha
tr
+ +
_
+ + +
-
tr
tr
+
C. brachycarpa
12. Keempferol 3-Rut
+ +
10. 5-Hydroxy-3,6,7,3',4',5'-hexamethoxyflavona
9. 5 , 4 ' - D i h y d r o x y - 3 , 6 , 7 , 8 , 3 ' - p e n t a m e t h o x y f l a v o n e
species
C. amplyocarpa
Cleome
CLEOMEAND CAPPARIS S P E C I E S
2. 5 , 7 , 4 ' - T r i h y d r o x y - 3 , 3 ' - d i m e t h o x y f l a v o n e
IN
tr
OF THE FLAVONOIDS
1.5,7,4'-Trihydroxy-3-methoxyflavone
Compound
T A B L E 1. D I S T R I B U T I O N
-
-
+ + +
-
+ +
+ +
-
-
-
-
+ + +
-
-
-
-
-
-
-
-
C. a/lilogenia
Capparis s p e c i e s
_
_
+
_
+ +
+
_
_
_
_
+
_
_
_
_
_
_
_
_
C. decidua
_
_
+
_
+
+++
_
_
_
_
÷+
_
_
_
_
_
_
_
_
C. spinosa
-I .I~o
~> z
o -11
¢n
< O z O
kaempferol, rhamnose kaempferol, glucose, rhamnose kaempferol, rhamnose kaempferol, glucose, rhamnose kaempferol, glucose, rhamnose quercetin, rhamnose quercetin, glucose, rhamnose quercetin, glucose, rhamnose quercetin, rhamnose quercetin, glucose, rhamnose isorhamnetin, glucose, rhamnose isorhamnetin, rhamnose unchanged
11 Kaempferol 7-Rha 12. Kaempferol 3-Rut 13. Kaempferol 3,7-diRha 14 Kaempferol 3-G-7-Rha 15. Kaempferol 3-Rha-7-G 16. Quercetin 7-Rha 17, Quercetin 3-Rut 18. Quercetin 7-Rut 19. Quercetin 3,7-diRha 20 Quercetin 3-G-7-Rha 21, Isorhamnetin 3-Rut 22. Isorhamnetin 3,7-diRha 23. Apigenin 6,8-di-C-G
G = Glucoside; Rha = rhamnoside; Rut = rutinoside
Complete acid hydrolysis
Compound kaempferol kaempferol, kaempferol 3-glucoside kaempferol 7-rhamnoside kaempferol, kaempferol 7-rhamnoside kaempferol, kaempferol 7-glucoside quercetin quercetin, quercetin 3-glucoside quercetin, quercetin 7-glucoside quercetin 7-rhamnoside quercetin, quereetin 7-rhamnoside isorhamnetin, isorhamnetin 3-glucoside isorhamnetin, isorhamnetin 7-rhamnoside unchanged
Mild acid hydrolysis
TABLE 2, CHEMICAL STUDIES OF THE ISOLATED FLAVONOID GLYCOSIDES
negative negative negative kaempfero! 7-rhamnoside kaempferol 3-rhamnoside negative negative negative negative quercetin 7-rhamnoside negative negative unchanged
Enzymatic hydrolysis
-rutinose rhamnose glucose glucose -rutinose rutinose rhamnose glucose rhamnose rhamnose unchanged
Hydrogen peroxide oxidation
----
-----
----
6.75 (s)--
13 14 15
16 17 18 19
20 21 22
23
(d, J= 2Hz) (d, J= 2Hz) (d, J= 2Hz) (s)
"See Table 1 for structures.
6.20(d,J=2Hz) 6.20(d,J=2Hz) 6.45 (d, J= 2 Hz)
6.20 5.20 6.30 6.45
6.45(d,J=2Hz) 5,45(d,J=2Hz) 5.48 (d, J= 2Hz)
6.40(d,J=2Hz) 6,20 (d, J=2Hz)
---
11 12
H-6
H-3
Compound no."
(d, J = 2 Hz) (d, J = 2 Hz) (d, J=2Hz) (s)
--
6.40(d,J=2Hz) 6.40(d,J=2Hz) 6,75 (d, J=2Hz)
6.42 6.40 5.45 6.75
6.82(d,J=2Hz) 6.77(d,J=2Hz) 6.77 (d, J = 2 Hz)
6.82(d,J=2Hz) 6.35 (d, J = 2 Hz)
H-8
(d, J=gHz) (m) (m) (m)
8.00 (m)
7.45(m) 7,80(s) 7.45 (m)
7.65 7.50 7.40 7.35
7.80(d.J=9Hz) 8.10(d,J=gHz) 8.10 (d, J=9Hz)
8.10(d,J=9Hz) 8.00 (d, J=9Hz)
H-2'
TABLE 3~ 1 H - N M R DATA OF THE ISOLATED FLAVONOID GLYCOSIDES
6.90 (d, J= 9 Hz)
----
-----
6.95(d,J=gHz) 6.90(d,J=9Hz) 6.90 (d, J = 9 Hz)
6.95(d,J=9Hz) 6.85 (d, J=9Hz)
H-3'
(d, J=9Hz) (d, J= 9 Hz) (d, J= 9 Hz) (d, J = 9 Hz)
6.90 (d, J = 9 Hz)
5.82(dd, J=9Hz) 6.90(dd, J=9Hz) 6.95 (d, J = 9 Hz)
6.90 6.82 5.80 6.93
6.95(d,J=gHz) 6.90(d,J=9Hz) 5,90 (d, J=9Hz)
6.95(d,J=9Hz) 6.85 (d, J= 9 Hz)
H-5'
8.00 (m)
7.45(m) 7.50(d,J=9Hz) 7.45 (m)
7.65 dd 7.50 (m) 7.40 (m) 7.35 (m)
7.80(d,J=gHz) 8.10(d,J=9Hz) 8.10 (d, J = 9 Hz)
8.10(d,J=9Hz) 8.00 (d, J= 9 Hz)
H-6'
5.53(s) 4.30 (d,J=2 Hz)
H-1 r h a m n o s e
1.1 (d,J=6Hz) 0.98 (d, J= 6 Hz)
CH3-rhamnose
4.20 4.20 4.50 5.60
(d, J = 2 Hz) (d, J=2Hz) (d, J= 2.5 Hz) (s), 5.40 (s)
0.90 (d, J = 6 Hz) 1.00 (d, J = 6 Hz) 0.95 (d, J = 6 Hz) 1,30, 0.95 (d, J= 6 Hz)
4,55, 5,20
--
--
5.15(d,J=7.5Hz) 4.40(s) 1.00(d,J=6Hz) 5.40(d,J=7Hz) 4.45(s) 0.99(d,J=SHz) -5.58, 5.40 (d, J= 2 Hz) 1.30, 0.95 (d, J= 6 Hz)
-5.15 (d, J = 7 Hz) 5.2 (d,J=7 Hz) --
-5.55,5.30(d,J=2Hz) 1.14,0.82(d,J=6Hz) 5.29(d,J=7.5Hz) 5.50(d,J=2Hz) 0.90(d,J=6Hz) 5.29 (d, J=7.5 Hz) 5.50 (d, J = 2 Hz) 0.90 (d, J= 6 Hz)
-5.30 (d, J = 7 Hz)
H-1 glucose
M. SHARAFETAL.
166
TABLE 4. MS DATA OF THE ISOLATED FLAVONOIDS GLYCOSIDES Compound no."
11
12
13
14
15
19
[M +1]+ [M+1-292]* M+ [M-308] [M + 1 - 162] ÷ [M + 1 - 308] + [ M - 162] + [M + 1 - 146] + [M+1-182] + [M+ 1-36] +
433 (47)
595 (18)
579 (22) 287 (46)
595 (3)
595 (35)
595 (1) 303 (35)
594 (100)
20
22
317 (27) 610 (55) 302 (2)
433 (34) 287 (3)
287 (100)
448 (13) 287 (46)
433 (47) 413 (8) 573 (7)
*See Table 1 for structures. FAB-mode was carried out for compounds 11 and 13, positive CI-mode for compounds 12, 14, 15, 19 and 22, while El-mode was carried out for compound 20.
indicated that compound 21 has a substitution at position 3, and at 3,7 positions for 22. Thus compound 22 is identified as isorhamnetin 3,7-dirhamnoside. H202 oxidation for 3 h of compound 21 afforded the disaccharide rutinose (Rt value, co-chromatography). Thus compound 21 is identified as isorhamnetin 3-rutinoside. 1H-NMR and MS supported the proposed structures (Tables 3 and 4). Finally, compound 23 is identified as apigenin 6,8-di-C-glucoside from its 1H-NMR and co-chromatography with an authentic sample. In conclusion, the chemistry of the four Cleome species and the three Capparis species investigated showed a clear difference in their flavonoid profile. Cleome species showed the presence of 10 methylated flavonoids and eight flavonol glycosides. On the other hand, only four flavonol glycosides were detected in Capparis species. Little has been reported on the flavonoids of members of the Capparaceae. Capparis spinosa is reported to contain rutin (quercetin 3-rutinoside) (Ahmed et al., 1972) as well as quercetin 7rutinoside (Artemeva et al., 1981). Both flavonoids were found to be present in the current study along with kaempferol 3-rutinoside and quercetin 3-glucoside-7-rhamnoside in C. spinosa. The present results appear to support Taeckholm's (1974) classification of Cleome under the family Cleomaceae. References Ahmed, Z. F., Rizk, A. M., Hammouda, F. M. and Seif-EI-Nasr, M. M. (1972) Glucosinolates of Egyptian Capparis species.Phytochemistry 11,251-256. Artemeva, M. V., Karryev, M. O., Meshcheryakov,A. A. and Gordienko,V. P. (1981 ) A new flavonol glycoside, quercetin 7-O-glucorhamnosidefrom Capparisspinosa. Izv. Akad. Nauk Turkm. SSSR, Set. Fizl- Tekh. 3, 123. Boulos, L. (1983) Medicinal Plants of North Africa, p. 52. ReferencePublications Inc., Algonac, Michigan. Greuter, W., Burdet, H. M. and Long, C. (eds) (1989) Med-Checklist. Conservatoireet Jardin botaniques, Geneva. Harborne, J. B. (1967) ComparativeBiochemistry of the Flavonoids. Academic Press, London. Mabberley, D. J. (1990) The PlantBook. Cambridge University Press,Cambridge. Mabry, T. J., Markham, K. R. and Thomas, M. B. (1970). The Systematic Identification of Flavonoids. Springer, Berlin. Markham, J. R. (1982) Techniquesof Flavonoid Identification. Academic Press,London. Sharaf, M., Mansour, R. M. A. and Saleh, N. A. M. (1992) Exudateflavonoids from aerial parts of four Cleome species. Biochem. Syst. Ecol. 20, 443-448. Taeckholm, V. (1974) Student Flora of Egypt, 2nd edn, pp. 167-169. CooperativePrinting Company, Beirut. Willis, J. C. (1966) A Dictionary of the Flowering Plants and Ferns. Cambridge University Press, Cambridge.