Pak. J. Bot., 35(2): 127-140, 2003. CHEMOTAXONOMIC STUDY OF INULA L. (S.STR.) AND ITS ALLIED GENERA (INULEAE - COMPOSITAE) FROM PAKISTAN AND KASHMIR RUBINA ABID AND M. QAISER Department of Botany, University of Karachi, Karachi-75270, Pakistan. Abstract Leaves of 21 taxa belonging to Inula L. (s.str.) and its allied genera viz., Pentanema Cass., Duhaldea DC., Dittrichia Greuter and Iphiona Cass., have been analyzed from Pakistan and Kashmir for their phenolic compounds. Analysis was carried out by two dimensional chromatography. Spectral analysis was performed on Shimadzu UV-spectrophotometer. The chemical data of this intricate group also support the generic delimitation of Dittrichia by having O-coumaric acid and Sakuranetin, while tricin 5-glucoside is exclusive for Iphiona and Duhaldea is distinctive by the presence of Vitexin and Isovitexin. However, Inula and Pentanema shared a mix pattern of compounds which points out the close relationship of both genera as compared to other genera of this group. Introduction There are several reports on the chemotaxonomy of the family Compositae. Most of the workers gave the attention to the flavonoids, sesquiterpene lactones and some other compounds (Crawford, 1970; Geissman & Irwin, 1973; Bohlmann et al., 1981; ValantVetschera & Wollenweber, 1981; Ates et al., 1982; Shukla & Gupta, 1985; Ling, 1992; Iwashina et al., 1995; Tzakao et al., 1995 and Akkal et al., 1997). Although some of the individual species of Inula and its allied genera were also chemically investigated but the chemical data was not utilized in the taxonomy of the genus. Dombrowicz & Greiner (1968) isolated quercetin and isoquercitrin from the aerial parts of Inula britannica L., Baruah et al., (1979) reported three new flavonoids from the aerial parts of Inula cappa (=Duhaldea cappa), Oksuz & Topcu (1987) reported a new Kaempferol derivative from the aerial parts of Inula britannica, Ahmed & Ismail (1991) isolated the sterols and a new flavonol from the aerial parts of Inula grantioides (=Iphiona grantioides). Oksuz & Topcu (1992) analyzed the extract of aerial parts of Inula graveolens (=Dittrichia graveolens) and isolated sesquiterpene lactones, flavonoids, dihydroflavonols and flavones. It is therefore evident from the preceding literature that some chemical informations are available on few taxa of Inula L., and its allied genera but no attempt has ever been made to utilize this data for taxonomical purposes. The present study was carried out to utilize the chemical data as a taxonomic evidence. Materials and Methods Leaves of 21 taxa belonging to Inula L. (s.str.) and its allied genera were analyzed for their phenolic compounds. For extraction, approximately 1 gm of dried leaves from herbarium specimens were extracted with 70% ethanol at room temperature. Extracts were concentrated and chromatographed two dimensionally on Whatman no.1 paper RUBINA ABID & M. QAISER 128 using two solvent combinations, i.e., BAW (n-butanol: acetic acid: water, 4:1:5) versus 15% acetic acid and BAW versus distilled water, following standard procedure of Harborne (1973). A list of voucher specimens is given in appendix-I. Phenolic compounds were identified by comparing with authentic markers along with the Rf values and colour in ultra-violet light before and after fuming with ammonia vapours. Compounds were repeatedly purified by paper chromatography, till the absorption properties became constant. Hence an elute of a paper blank in 95% ethanol (usually about 150cm2) was taken and applied (spotted) to the paper, and run in BAW and 15% HOAc separately. After the purification of compounds, the spots of chromatogram were cut and shaken in 95% ethanol for 30 minutes. The solution was filtered and allowed to concentrate, and directly used for spectral analyses on Shimadzu UV-240 spectrophotometer. Results and Discussion Paper chromatography of the aqueous ethanolic extracts from the leaves of 21 taxa of Inula L., and its allied genera led to the isolation of 42 (including 11 unknown compounds) different phenolic compounds (phenolic acids, flavonols, flavones, glycosylflavones, flavanones and chalcones) (Table 1.1-1.5, Fig. 1.1-1.7). Generic key based on chemical characters 1. + - Vitexin and iso-vitexin both present ......................…………………… Duhaldea No such compounds present ...........................................……………………….. 2 2. + - Tricin 5-glucoside present ...................................……………………….. Iphiona Tricin 5-glucoside absent ...............................................……………………….. 3 3. + - Sakuranetin present ....................................................………………… Dittrichia Sakuranetin absent ........................………………….………... Inula, Pentanema The results suggested that the chemical data of flavonoids is not always correlated with the classification. This contention was also supported by Heywood (1973), as he pointed out that the flavonoids themselves could not be taken as independent evidence relative to supporting a particular classification. However, it may be useful in taxonomic delimitation at generic or specific level. Iphiona and Dittrichia are chemically related to Inula (s.str.) as all of these genera share a number of flavonoids, although the presence of O-coumaric acid along with sakuranetin in Dittrichia, and tricin 5-glucoside in Iphiona, keep them distinct from the other genera. Duhaldea is chemically separated from the other genera due to the occurrence of vitexin, iso-vitexin and quercetin 4-glucoside. The flavonoid patterns of the genera Inula (s.str.) and Pentanema are similar to the other genera as they share number of flavonoids and do not possess any specific compounds of their own. Although the presence of certain specific compounds in other genera, make them separate. However, the rarer occurrence of ferulic acid, aesculetin, aesculin, some glycosides of quercetin, kaempferol, luteolin and hesperitin in Inula (s.str.) further make it distinct from all the other genera. CHEMOTAXONOMIC STUDY OF INULA AND ITS ALLIED GENERA 129 130 RUBINA ABID & M. QAISER CHEMOTAXONOMIC STUDY OF INULA AND ITS ALLIED GENERA 131 132 RUBINA ABID & M. QAISER Table 1.4. Total number of flavonol, flavone glycoside and c-glycosyl flavones in Inula L. (s.str.) and its related genera. Total no. of compounds Name of genera Flavonol Flavone C-glycosyl glycosides glycosides flavones 32 12 5 Inula 9 2 Pentanema 4 2 7 Duhaldea 3 1 Dittrichia 3 2 Iphiona Table 1.5. Unidentified compounds of Inula L. (s.str.) and its related genera. Rf values Colour in UV Name of taxa BAW 15% With Without HOAc ammonia ammonia 54.96 74.40 l. yell. yell. br. Inula koelzii 60.02 99.33 yell.gr. yell. gr. 55.38 74.84 l. yell. Yell. br. I. royleana 62.92 100.02 yell.gr. yell. gr. 0.0 92.0 b.bl. l. bl. I. racemosa 19.38 97.74 b. purp. purp. bl. 61.88 99.81 yell. gr. yell. gr. 22.22 100.0 b. purp. purp. bl. I. stewartii 0.0 97.50 d. br. d. br. 92.91 86.90 Yell. Yell. I. orientalis 28.05 17.20 b. yell. b. yell. I. clarkei 94.28 86.88 yell. yell. I. obtusifolia 95.66 87.05 yell. yell. I. britannica 20.50 45.25 bl. bl. I. acuminata I. falconeri 70.75 26.30 b. purp. purp. bl. I. rhizocephala 19.28 39.99 bl. bl. 0.0 91.66 b. bl. l. bl. Pentanema glanduligerum 24.80 99.87 b. purp. purp. bl. 19.99 98.20 b. purp. purp. bl. P. indicum P. divaricatum P. vestitum 59.25 93.06 b. bl. l. bl. Duhaldea cappa D. eupatorioides D. cuspidata 69.21 92.42 yell. d. yell. Dittrichia graveolens Iphiona aucheri 82.05 99.12 yell. yell. gr. I. grantioides Key: yell. = yellow; gr. = green; br. = brown; purp. = purple; bl. = blue; b. = bright; d. = dull; l. = light. CHEMOTAXONOMIC STUDY OF INULA AND ITS ALLIED GENERA 133 Fig. 1.1. Absorption spectra of Caffeic acid (curve A) and Chlorogenic acid (curve B) in 95% EtOH. Fig. 1.2. Absorption spectra of Ferulic acid (curve A, marker; curve B, isolated compound) in 95% EtOH. Fig. 1.3. Absorption spectra of Aesculetin (curve A) and Aesculin (curve B) in 95% EtOH. 134 RUBINA ABID & M. QAISER Fig. 1.4. Absorption spectra of Rutin (curve A, marker; curve B isolated compound) and Quercetagetin (curve C) in 95% EtOH. Fig. 1.5. Absorption spectra of Luteolin 7-glucoside (curve A) and Kaempferol 3-sophorotrioside 7rhamnoside (curve B) in 95% EtOH. CHEMOTAXONOMIC STUDY OF INULA AND ITS ALLIED GENERA 135 Fig. 1.6. Absorption spectra of Apigenin 7-glucoside (curve A, marker; curve B isolated compound) in 95% EtOH. Fig. 1.7. Absorption spectra of Isoliquiriteginin 4-glucoside (curve A) and Butein 4-glucoside (curve B) in 95% EtOH. RUBINA ABID & M. QAISER 136 Key to the species of Inula (s.str.) based on chemical characters 1. + - Kaempferol 3-sophoroside 7-rhamnoside and kaempferol 3-sophorotrioside 7rhamnoside both present ........................……………………………….. I. koelzii No such compounds present ...........................................……………………….. 2 2. + - Iso-orientin present ..........................................………………………… I. clarkei Iso-orientin absent .........................................……………………… I. obtusifolia 3. + - Kaempferol 3-lathyroside 7-rhamnoside and luteolin 7-glucoside both present. ………………………………………................................................................... 4 Both the compounds absent ……………………............................................…. 5 4. + - Kaempferol 3-sophoroside 7-rhamnoside present ……………...…... I. orientalis No such compound present ............................……………………... I. britannica 5. + - Butein 4-glucoside present ................ I. acuminata, I. falconeri, I. rhizocephala Butein 4-glucoside absent ..............................................……………………….. 6 6. + - Quercetagetin present …............................................………………... I. stewartii Quercetagetin absent ........................................................……………………… 7 7. + Apigenin 7-glucoside and apigenin 8-rhamnosyl glucosyl both present ……... ………………………………………………………………………... I. royleana Both the compounds absent ...............................……………………. I. racemosa - In the genus Inula L. (s.str.), I. koelzii Dawar & Qaiser, I. royleana DC., I. racemosa Hook.f., and I. stewartii R. Abid & Qaiser are generally related in their morphology as they have upper sessile and semiamplexicaul, while lower winged petiolate leaves and 3-4 mm long and usually glabrous cypselas, similarly, the chemical data of these species also strengthen the above morphological correlation due to the presence of iso-orientin, kaempferol 3-sophorotrioside 7-rhamnoside and caffeic acid. Some other glycosides of kaempferol, quercetin, apigenin, luteolin and tricin are absent from all the four species. However, the four species can still be differentiated on the basis of flavonoid pattern, I. koelzii and I. stewartii have aesculetin but lacks in I. racemosa and I. royleana, while I. koelzii and I. stewartii differ from each other by the presence of ferulic acid and rutin respectively. On the other hand, I. royleana and I. racemosa differ due to occurrence of apigenin glucoside in I. royleana and hesperidin in I. racemosa respectively. Inula britannica L., I. acuminata Royle ex DC., and Inula falconeri Hook.f., are more or less morphologically related species which share several compounds viz., rutin, quercetagetin and apigenin 6-OH but I. britannica can be delimited from both the species by the presence of kaempferol 3-lathyroside 7-rhamnoside, luteolin 7-glucoside and isoliquiritigenin 4glucoside. I. acuminata and I. falconeri could not be distinguished chemically from each other and share all the compounds with the exception of an unidentified compound with Rf values 20.5 (BAW), 45.25 (15% HOAc) and with unchanged blue colour, that is exclusive for I. acuminata. On the other hand, the specific compounds of I. britannica are also shared with I. orientalis Lamk., although both are morphologically dissimilar furthermore, both the species are also delimited due to the occurrence of kaempferol 3-sophoroside 7-rhamnoside CHEMOTAXONOMIC STUDY OF INULA AND ITS ALLIED GENERA 137 in I. orientalis and apigenin 6-OH in I. britannica. Likewise, Inula obtusifolia Kern., and Inula clarkei (Hook.f.) Stewart are also closely related species as they share ferulic acid, caffeic acid, quercetin 3-sophoroside 7-glucoside, quercetagetin, kaempferol 3-rutinoside 7glucuronide and apigenin 8-rhamnosyl glucosyl. However, these 2 species can be differentiated from each other as the quercetin aglycon and iso-orientin are present in I. obtusifolia and I. clarkei respectively. However, Inula rhizocephala Schrenk is morphologically distinct from rest of the species due to stemless habit, similarly the presence of aesculin in I. rhizocephala also makes it chemically distinct from rest of the species of Inula L. Key to the species of Pentanema based on chemical characters 1. + - Rutin present ............................................................…………………………… 2 Rutin absent ..........................................………………………. P. glanduligerum 2. + - Quercetagetin present …………………………................................................... 3 Quercetagetin absent .......................................………………………. P. indicum 3. + Apigenin 7-glucoside and kaempferol 3-sophorotrioside 7-rhamnoside both present .......................................…………………………………. P. divaricatum No such compounds present ……...............................………………. P. vestitum - Species of Pentanema Cass., could be delimited on the basis of flavonoid pattern. On the other hand morphologically unrelated species share the similar compounds for instance, kaempferol 3-sophorotrioside 7-rhamnoside is present in 2 morphologically different species viz., Pentanema glanduligerum (Krasch.) Gorschk., and Pentanema divaricatum Cass. Similarly apigenin 7-glucoside is found in Pentanema indicum (L.) Ling and P. divaricatum both of them are morphologically dissimilar to some extent. However P. glanduligerum is distinguished from all of the other species of Pentanema by having the quercetin aglycone while other 3 species have rutin. P. vestitum (Wall. ex DC.) Ling and P. divaricatum are separated from P. indicum by the presence of caffeic acid and quercetagetin but absent from P. indicum. P. divaricatum is splitted from P. vestitum due to the presence of apigenin 7glucoside and kaempferol 3-sophorotrioside 7-rhamnoside which are exclusive for P. divaricatum. Key to the species of Duhaldea based on chemical characters 1. + - Apigenin 7-glucoside and iso-vitexin both present ...............…………………... 2 No such compounds present ........................…………………... D. eupatorioides 2. + - Quercetagetin present ...................................………………………. D. cuspidata Quercetagetin absent ..........................................………………………. D. cappa Chemical variation is not so pronounced within all the species of Duhaldea DC., as Duhaldea eupatorioides (Wall.ex DC.) A.Anderb., shares all its compounds with D. cuspidata (Wall.ex DC.) A.Anderb., and no specific compound is found in D. eupatorioides. However, the D. cuspidata can be separated from D. cappa (Ham.ex D.Don) A.Anderb., due to the occurrence of chlorogenic acid and quercetagetin which are exclusive for D. cuspidata. 138 RUBINA ABID & M. QAISER Key to the species of Iphiona based on chemical characters 1. + - Quercetagetin present ..................................………………………. I. grantioides Quercetagetin absent ..........................................……………………… I. aucheri Both the species of Iphiona Cass., are chemically distinguished from each other due to the presence of quercetagetin in I. grantioides (Boiss.) A. Anderb., but absent from I. aucheri (Boiss.) A. Anderb. Apart from the known compounds several unidentified compounds are also found within the genus Inula L. (s.str.) and its allied genera (Table 1.5). Those spots which have more or less same Rf-values and colours are tentatively grouped in 11 different compounds. Morphologically dissimilar taxa share same unknown compounds. For instance a compound with Rf-values 0.0 and 91.66-94.93 (blue coloured) respectively in BAW and 15% HOAc is common in Inula racemosa and Pentanema glanduligerum. Another compound which is purplish blue in colour having the Rf-values from 19.38-24.8 (BAW) and 97.74-100 (15% HOAc) is found in Inula stewartii, I. racemosa, Pentanema glanduligerum and Pentanema indicum. Inula rhizocephala and Inula acuminata also share a compound, which has blue colour with Rf-values from 19.28-20.5 (BAW) and 39.99-45.25 (15% HOAc). On the other hand Dittrichia graveolens (L.) Greuter has an exclusive unknown compound with the Rfvalues 69.21 (BAW) and 92.42 (15% HOAc) (colour change from yellow to dull yellow after ammonia fumigation). Similarly, Inula rhizocephala has an exclusive unknown compound with purplish shade and 70.75 (BAW) and 26.30 (15% HOAc) Rf-values. However, Inula clarkei and I. obtusifolia are closely related but an unknown compound with the Rf-values 28.05 (BAW) and 17.20 (15% HOAc) with bright yellow colour is found only in I. clarkei. Likewise, Iphiona grantioides has an unknown compound which is yellow / yellowish green in colour with the Rf-values 82.05 and 99.12 in BAW and 15% HOAc respectively is not shared by closely related taxon Iphiona aucheri. It is generally agreed that flavonol glycosides (kaempferol, quercetin, particularly myricetin) are present in the supposedly more primitive dicotyledons and flavone oglycosides (apigenin and luteolin) occur in more highly advanced dicotyledons, c-glycosyl flavones (vitexin, iso-vitexin, orientin and iso-orientin) are considered as an intermediate state between flavonols and flavone o-glycosides (William & Harborne, 1971; Harborne, 1977; Crawford, 1978; Omer et al., 1996). The present studies are also in accordance with this contention. All the genera have shared a mix pattern of flavonol, flavone o-glycoside and c-glycosyl flavone. Although the flavonols predominated over flavone. Likewise, Inula L. (s.str.) shared some morphologically primitive and advance characters so it is paraphyletic in origin and this paraphyletic condition of the genus was earlier reported by Anderberg (1991). However, primitive characters are quite dominant in this genus with always radiate capitula and pappus bristles in large number, while on the other hand it is also characterized by the presence of herbaceous nature of plants and conspicuously ribbed cypselas. So this morphological evolution in Inula (s.str.) also support the above mentioned generalizations as in this genus flavonol glycosides predominated over flavone glycosides by the presence of 32 flavonol glycosides and 10 flavone glycosides and the number of flavonol gradually reduced to 9, 4, 3, and 3 in the remaining genera viz., Pentanema, Duhaldea, Dittrichia and Iphiona respectively (Table 1.4). This clearly indicated gradual co-evolution of flavonoids and morphological characters. This predominant pattern of flavonols over flavones was also observed by Seelingmann (1996) in certain tribes of Compositae. The mix pattern of flavonol, flavone o-glycoside and c-glycosyl flavone in all the genera is also in agreement with a number of instances given by Crawford (1978) and Averett et al., (1986) in which all three types (flavonols, flavone o-glycoside and c-glycosyl flavones) have been observed. CHEMOTAXONOMIC STUDY OF INULA AND ITS ALLIED GENERA No. 1. 2. Taxon Inula koelzii I. royleana 3. I. racemosa 4. 5. I. stewartii I. orientalis 6. I. clarkei 7. I. obtusifolia 8. 9. 10. I. britannica I. acuminata I. falconeri 11. I. rhizocephala 12. Pentanema glanduligerum 13. P. indicum 14. P. divaricatum 15. P. vestitum 16. Duhaldea cappa 17. 18. D. eupatorioides D. cuspidata 19. 20. Dittrichia graveolens Iphiona aucheri 21. I. grantioides 139 Appendix-I Collector, number and herbarium W. Koelz 2900a (KUH); W. Koelz 2827a (NY) Y. Nasir & Rubina Akhtar 12996 (RAW); M. Qaiser & Rizwan Y. Hashmi 7868 (KUH) R.R. Stewart 14052 (KUH); R.R. Stewart 19550 (RAW) R.R. Stewart s.n. (RAW) S. Abedin & M. Qaiser 8887 (KUH); Tahir Ali, M. Qaiser & M. Ajmal 503 (KUH). Hans Hartmann s.n. (RAW); E. Nasir & G.L. Webster 5804 (RAW) M. Qaiser, S. Omer & S.Z. Hussain 8414 (KUH); R.R. Stewart 18803 (RAW) R.R. Stewart 54 (RAW) Stainton 3077 (RAW); R.R. Stewart 26356 (RAW) R.R. Stewart 20484 (KUH); M.A. Siddiqui, Y. Naisr & Zaffar 4182 (K) R.R. Stewart 18859 (RAW); S. Omer & M. Qaiser 2360 (KUH) G.R. Sarwar & S. Omer 256 (KUH); Stainton 2944 (RAW) A. Rashid 26985 (RAW); Farrukh Hussain s.n. (RAW) S. Abedin & Abrar Hussain 6232 (KUH); S.M.H. Jafri 2854 (KUH) Y. Nasir & Rubina Akhter 11863 (RAW); S. Abedin 2659 (KUH) A. Ghafoor & Tahir Ali 4005 (KUH); S.A. Farooqui & M. Qaiser 3172 (KUH) R.R. Stewart & I.D. Stewart 4145 (RAW) Tahir Ali, M. Qaiser & M. Ajmal 367 (KUH); Y. Nasir & Nazir 10519 (RAW) J.L. Stewart 245 (K) Tahir Ali & G.R. Sarwar 2868 (KUH); Tahir Ali 1478 (KUH) S. Omer & Rizwan Y. Hashmi 2003 (KUH); A. Ghafoor & S. Omer 1825 (KUH) References Ahmed, U.V. and N. Ismail. 1991. 5-Hydroxy-3, 6, 7, 2’, 5’-Pentamethoxyflavone from Inula grantioides. Phytochem., 30(3): 1040-1041. Akkal, S., F. Benayache, S. Benayache. and M. Jay. 1997. Flavoniods from Centaurea incana (Asteraceae). Biochem. Syst. & Eco., 25(4): 61-362. Anderberg, A. 1991. Taxonomy and phylogeny of tribe Inuleae (Asteraceae). Pl. Syst. Evol., 176: 75-123. Ates, N., A. 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