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Sesamin, sesamolin and the origin of sesame
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Sesamin, Sesamolin and the Origin of Sesame DOROTHEA BEDIGIAN*, DAVID S. SEIGLERI" and JACK R. HARLAN* *Crop Evolution Laboratory, Deparlment of Agronomy, University of Illinois, 1102S. Goodwin, Urbane, IL 61801, U.S~.; tDepartment of Plant Biology, University of Illinois, 505 S. Goodwin, Urbena, IL 61801, U.S~.
Kay Wind Index--Ses~rnurn/rid/cure; Pedaliaceae;lignan; phenylpropanoid; sesamin; =esamolln; TLC; NMR; evolution. Abslmct---Cultivars of sesame were screened to determine how widely lignens Occur. All lines tested contained sesamin and sesamolin. Sesamin and lesamolin in other species of Sesamumvaried. Some other genera in the Pedaliaceaealso possessed lignans. Phylochemical evidence as well as other data support the suggestion that the progenitor of sesame occurs in india.
Introduction The wild progenitor of sesame, Sesamum indicure L has not been established with certainty and the literature asserts with equal frequency that the center of origin of the crop is in Africa and Asia [1]. A recent proposal that Sesamum la~fo/ium Gillett is the progenitor of sesame and that the crop originated in Africa was accompanied by a division of the genus Sesamum into four sections [2]. The Pedaliaceae, to which sesame belongs, have been considered related most closely to the Bignoniaceae, Martyniaceae and Acanthaceae [3-5]. Although the use of chemical data to establish phylogenetic relationships is important [6-8], lignans have not been widely used for these analyses. Lignans are widespread in the plant kingdom [9, 10], occurring in heartwood, leaves and resinous exudates of plant families as distantly related as the Gingkoaceae and the Asteraceae. Approximately three dozen compounds are known at present [10]. Relationships of members of several families including the Asteraceae [11-13] have been studied with lignans but these compounds have not been used to study the phylogeny of plant families related to the Pedaliaceae. Seeds of sesame contain the lignans sesamin and sesamolin. These compounds are not found in other edible oils. The effects of strain, location grown, aging and frost damage on the sesamin, sesamolin and sesamol content of sesame seed (/bce/t~d~ &o~/lS84) 133
have been examined [14]. In a related species, Sesamum angolense Welw., the isolation and structure of a novel compound, sesangolin, an insecticidal synergist, has been reported [15]. The presence of sesamin was not reported. These workers obtained the oil from Pearman eta/. [16], who had submitted a seed sample to the director of the Royal Botanic Gardens, Kew, for identification; the seeds were believed to be those of S. ango/ense. Oil from the same seeds was reported to contain sesemin by Pearman eta/. [16]. The families considered to be the nearest relatives of the Pedaliaceae, the Bignoniaceae and the Acanthaceae, also contain lignans. Sesamin as well as a new lignan, peulownin ware isolated from the wood of Pau/ownia tomentosa Staud. (Bignoniaceae) [17, 18]; NMR spectral analysis of the stereochemistry of the latter compound shows structural similarity to sesamin and sesamoiin. Roots and stems of Phy//arthron comorense and Tabebuia rosea respectively (Bignoniaceae) yielded sesamin and peulownin [19]. The lignans sesamin, asarinin and sesamolin and a new compound, simplexoiin, were isolated from Just~'cia $imp/ex (Acanthaceae) [20]. Recent reports of sesamin in other related families include Hyp~'s tomentosa (Lamiaceae) [21], Aptos/mum spinescens Thunbg. (Scmphulariaceae) [22] and Gme/ina arborea Roxb. (Verbenaceae) [23, 24]. Other taxa from which sesamin and sesamolin have been reported are Acan~hopanax sentJcosus [25] and A. sess/liflorum (Araliaceae) [26]; Asarum sieboldii (Aristolochiaceae) [9, 27]; Anacyclus
134
pyrethrum (Asterceae) [28]; Artemisia absinthium group [12, 13, 29, 30]; Chrysanthemum cinerariaefo/ium (Asteraceae) [31]; C. frutescens [32]; C. indicum (Asteraceae) [33]; Diotis rnari~'ma (Asteraceae) [34]; Ageratina, Critoniaand Fleischmannia (Asteraceae), formerly part of the genus Eupatorium, [35]; Otanthus maritimus (Asteraceae) [36, 37]; Alnus g/u#'nosa (Betulaceee) [38]; Salicomia europaea (Chenopodiaceee) [39]; Austrocedrus chi/ensis (Cupressaceee) [40]; CharnaecypaHs (Cupressaceee) (a garden species that has hibalactone which upon reduction yields hinokinic acid and d-sesamin) [41]; Gingko biloba (Gingkoaceae) [42, 43]; Machi/usglaucescens(Lauraceee) [44, 45]; Ocotea usarnbarensis (Lauraceee) [46]; species of Magno/ia (Magnoliaceae) [47-49]; Talauma hodgsonii (Magnoliaceae) [50]; Picea abies (Pinaceee) [38]; Macropiper exce/surn (Piperaceee) [51]; the genus Piper (Piperaceee) [52-58]; Evodia micrococca var. pubescens (Rutaceae) [27]; Fagaraxanthoxyloides(Rutaceae) [59]; F/indersia pubescens (Rutaceae) [60]; and the genus Zanthoxylum (Rutaceee) [61-66]. Lignans are active constituents of certain medicinal plants [10]. Podophyllin, a complex resinous extract of may apple, Podophyllum peltaturn, has been used as a powerful cathartic. The principal lignan constituent, podophyllotoxin, is of interest as it has cytotoxic action similar to colchicineo Podophyllotoxin and other lignans that possess the partially reduced naphthalene nucleus have shown some promise in treating certain types of neoplasms [10, 67]. However, when constituents of Hyptis tomentosa (Lamiaceae) were screened for anticancer agents, sesamin was found to be inactive [21]. It has been reported that sesamin and sesamolin may play a role in natural seed dormancy. Germination inhibition by sesamin and sesamolin is considerably greater with peanut and cucumber seeds than with rice, where only retarded coleoptile growth occurred [68]. The effect is more pronounced on lipid-storing seeds than on seeds containing carbohydrate as storage product, and it has been suggested that the effect is on the end product initiating enzymes controlling lipid mobilization [68]. A germination inhibitor isolated from Aegilops ova~a [69] bears a close structural resemblance to sesame lignans. Leached hulls inhibited the germination of Lactuca achenes. Inhibition was
DOROTHEABEDIGIAN,DAVIDS. SEIGLERAND JACK R. HARLAN
considerably stronger in light than in darkness. The authors claimed that it was a lactone. This report was questioned [24] and the compound later shown to be the lignan acanthotoxin [70]. This compound is also found in Zanthoxylum acanthopodium (Rutaceae) and has spectral properties identical with those of justicidin E, isolated from Justicia procurnbens (Acanthaceae) [70]. Extracts of wild sesame seed from India inhibited seedling growth of cultivated sesame [71]. Root exudates of sesame are repellant to root knot nematodes of eggplant, tomato, potato and okra [72, 73]. Sesame was as potent a nematicide as marigold. Field trials showed that intercropping with sesame is economically beneficial as the nematode problem is eliminated and in addition the sesame can be harvested. The action of sesamin and sesamolin as inhibitom of mixed function oxidases [74] is due to the methylenedioxyphenyl group. This group has been recognized previously as an effective inhibitor of microsomal oxidation. Sesamin and sesamolin have been used for practical applications as antioxidants and insecticides. In the course of testing pyrethrum extracts in combination with a number of vegetable and fish oils, it was found that sesame oil, but not others, markedly increased the effectiveness of pyrethrum insecticides [75]. Sesamin and sesamolin enable sesame oil to resist oxidative rancidity [76-78], an action that is also attributed to the methylenedioxyphenyl group. Sesamin and sesamolin are used as active ingredients in antioxidants, antiseptics, bactericides, viricides, disinfectants, moth repellants and anti-tubercular agents. These uses are mentioned in a series of patents. Sesame oil has been used as a control in pharmacological experiments. We undertook this study to learn more about the phylogenetic relationships of sesame and the Pedaliaceae. We were seeking new data to help ascertain the origin of the cultivated crop and to learn the extent to which sesemin and sesamolin occur in related taxa. Neighboring species, genera and families were examined. We were also interested in a practical procedure for the determination of sesamin and sesemolin by TLC to screen seed oils of members of this group. We attempted to develop an analytical method to
135
SESAMIN, SESAMOLIN AND THE OffiGIN OF SESAME
screen oils rapidly as well as to isolate and identify sesamin and sesemolin with NMR spectropscopy in order to determine structure and to assess purity. Results Oils of the taxa studied were separated on silica gel by TLC or column chromatography. Sesamin and sesamolin ware resolved from other seed oil compounds. We also defined a solvent system for TLC that would separate the lignans of sesame. After removing triglycerides, the best separation was obtained with a chloroform, benzene and MeOH mixture (60:40:1). Sesamolin travelled nearest the solvent front (Rf 0.54) and sesemin lagged behind it (Rf0.36). Up to six other spots ware observed under u.v. light after spraying the plates with 2',7'-dichlorofiuorescein, or after acid charring, but these ware found to be non-lignan lipids by NMR spectroscopy. The first step in the taxonomic comparisons was to examine 50 cultivars of sesame that originated in different geographic regions of the world. We found no significant differences in sesamin and sesemolin content among the cultivers. All samples tested possessed both lignans. As no lines of sesame contained sesamol, a hydrolysis product [77] of sesamolin, it did not appear that our procedure was accompanied by hydrolytic degradation. We attempted to locate where in the seed the lignans are stored. Dissection of seeds to remove the oily endosperm that wraps around the embryo proved to be difficult and contamination of the embryo fraction with some endosperm tissue was unavoidable. Our results show that the embryo fraction lacks sesemolin, which is associated only with endosperm tissue. Sesamin was found in both portions. We also examined near relatives of cultivated sesame to see how widespread these lignans are in the genus Sesemum (Table 1). Sesamin and sesemolin were found in seed oils of S. angolense, S. angustJfolium, S. calycinum, S. indicum, S. offentale var. malabaricum and a weedy escape from Australia. Oils of $. laUfoliurn and S. radiatum contained sesemin but not sesamolin. The presence of lignans in other species was more difficult to establish. These compounds were absent from fresh seed of Sesarnum alatum. Even when relatively large amounts of seed were
TABLE 1. UGNAN CONTENT OF SEEDS OF SPECIES OF SESAMUM Species
Seed
S~samin
Selamolin
$~s~mum a/a~urn Thonn. & engo/en~e Welw. ~ a n ~ m ( Q ~ . ) E~I. ~ ~um Welw. ~ ~ Bu~.
f h f h h
-+ + + -
-+ + + T~ce
~ ~um
L.
f
+
+
~ ~ ~ ~
Gi~ ~r. ~ ~ . ~um.
f f h f
+ + T~e +
-+ -
h h
---
---
f
+
+
~ m ~ ~ ~
~r. ex. H ~ m ~ ~ .
S~m~.~. ~ m Ihl. ~ ~ i . ~ ~ m We~. ex. ~ . U. ~ I. No. ~ : ~ ~ ~ ~lia.
Seed source code: f--fresh seed; h-herbarium specimen. Voucher specimen numbem given in Experimer,~al section.
extracted as described below, the sample contained only non-lignan lipid$ as indicated by NMR spectral analysis. TLC also confirmed that lignans were absent. In this study, seeds of Sesemum angolense from herbarium specimens showed the presence of both sesamin and sesamolin. A spot identical in Rf to sesamin was observed; we cannot exclude the possibility, however, that the compound is sesangolin, not sesamin. Confirmation with fresh seed material and NMR spectroscopy could resolve this problem. The use of TLC of lignans as a diagnostic tool can be illustrated by the following example. The germplasm collection maintained by the Agricultural Research Corporation, Kadugli, Sudan, contains line 144 that was identified as Sesamum indicum. The shape and color of the corolla, and overall plant habit, and the fact that this cultivar remained green well after all other lines in the nursery had begun to senesce suggested that it differed greatly from other cultivars of sesame. It was the last line to be harvested. Testa ornamentation on seeds from line 144 appeared similar to those of specimens of Sesarnurn radiaturn (D. Bedigian, unpublished data). Extracts of both samples were compared by TLC. The chromatograms were identical and it is probable that line 144 is actually S. radiatum. Other genera of the family Pedaliaceae ware examined to determine the extent to which they
136
DOROTHEA BEDtGIAN, DAVID S. SEIGLER AND JACK R. HARLAN
TABLE 2, UGNAN CONTENT OF SEEDS OF RELATED GENERA OF SESAMUM Species Ceratotheca sesa~ide$ Endl. Cemmff~va 1~7obaMeyer ex Bemh. Ho/ub~ s~cca= Oily. Pedalium murex L. P~t~e z~nguebericum Gay Ptetodiscus aur~n~cus Welw. P.o~en~~termphy//~ Gay ex Del. ~ff~mnus ~lse~nu$ Engl.
Seed
Sesamin
Sesamolin
f h h h h h f h
÷ Trace Trace Trace Trace ÷
Trace -----+
contain lignans (Table 2). These genera vary considerably in their content of lignans. The presence of lignans in plant families related to the Pedaliacaae was also examined. Fresh seeds of the taxa listed (Table 3) were studied (nomenclature according to Hortus Third) [79]. Sesemin and sesemolin were not found in the seed oils of representatives of other plant families that we tested. Several of these did contain a similar sedes of non-lignan lipids, but NMR studies showed that those compounds were not lignans. Although traces of other lignans could be present, they are not major components as they are in sesame. The wild relatives of sesame, including S. latifol/um, have dormant seeds; this was confirmed by Sudanese agronomists (M. O. Khidir and M. A. Mahmoud, pers. comm.). Dormancy of both S. latifoliurn and S. orientMe var. malabaricum was broken only by leaching the seeds overnight in running tap water, followed by 3% hydrogen peroxide soaks. Germination was still only 20% (D. Bedigian, unpublished date). TABLE 3. TAXA STUDIED IN PLANT FAMILIES RELATED TO THE PEDALIACEAE
Acanthus rnollis L. (Acanthaceae) Ca=/pa b/geoe/o/de~Walt. (Bignoni~ceae) Peu/oww/e mmento=a ('rhunb.) St~ud. (Bidnoniaceae) Bor~go o f f l ~ / ~ L. (Boraginaceae) Omphalo#es I~ifoli~ (L.) Moench (Borlginaceae) Coleus x hyt~du= VOLS. (Lamlaceae) S~h,~ ver//cB~= L. vat. ~tba (Lamiaceae) Pmbosc/de~ ~ (MB.) TheN. (Mortynioceae) from Arizoea ~ ~ (Mill.! The¢. ~ l from O~dahoma Digi~li$ ,ou,-pureaL. (S~-.7#~d~riaceae) Mk~u/us ~ Dougl. ex Benth. ( S c ~ u l l r i a c e a e ) PenBtemon c.~ml~nubtus (C~v.) Willd, (Scrol~hulari=ceae) Torenia vio~cea (Azaola) Penn. (Scrophularlaceae) C.len~endrum ~ Dode ~ = e ) /.~nCanac~m,~r#L. Nedoenaceoe)
Discussion We have been able to confirm the identity of two lignans, sesamin and sesamolin in sesame oil. These are recognizable under UV light on TLC plates, but their identification was also established by NMR spectroscopy. By the same methods, we have shown that S. alarum consistently lacks these constituents. Sesangolin does not appear to be present in S. ango/ense. Although we cannot exclude the presence of other lignans, we saw no evidence for the presence of this compound, as the lignans of S. angolense had the same Rt values as those of sesame. As the materials previously studied were apparently not supported by voucher specimens (none is cited), we cannot be sure of the identification of the plant examined. Additionally this report appeared prior to the description of S. latifolium [80], a species with which S. angolense might easily be confused and this makes the identification even more tentative. The absence of lignans from both S. alarum and S. capense is noteworthy as both are species with winged seeds in the section Sesamoptaris, that is relatively isolated within the family [2]. Annotations on herbarium specimens show that there is disagreement concerning the identification and synonomy of these two species (D. Bedigian, unpublished data). Although sesamin and sesamolin are reported to occur in members of related families, investigation of a series of plants from these families, chosen at random, did not reveal sesamin and sesamolin in families other than the Pedaliaceae. As mentioned previously, Sesamum latifolium has been suggested as the ancestor of sesame [2]. This species occurs in East Africa and contrary .to previous reports, is widely distributed throughout the Sudan [81]. The chromosome number of S. latifolium is 2n-32 [2]. Reciprocal crosses between the crop and S. latifolium have been attempted extensively by Sudanese breeders (M. O. Khidir, pers. comm.; D. Bedigian, unpublished data), but have never succeeded. Only a few shrunken seeds produced in a few tiny twisted capsules were obtained. Attempts to germinate these seeds were futile (D. Bedigian, unpublished data). Samples of seeds of S. /atJfolium consistently lack sesamolin. We regard the nearest wild relative of cultivated sesame as the taxon known in India as the
SESAMIN, SESAMOLIN AND THE ORIGINOF SESAME
137
wild gingelly of Malabar, named Sesamum orientale var. malabaHcum Nar. in the original description [82]. This taxon is the probable ancestor of the crop and like cultivated sesame, contains both sesamin and sesamolin. Numerical analyses of the patterns of morphological variation in sesame [83] confirm the nearness of relationship between this wild Indian variety and the crop. The closeness of the relationship between the crop and its putative ancestor is also shown by reciprocal crosses, which were fully ferlJle using ver. rnalabaHcurn as both male and female parent. The chromosome number of S. orien~ale vat. malabaHcum is 2n-26, the same as the crop [82]. As no Latin description was given atthe time of publication [82], the report provides information about the plant but does not constitute a valid taxonomic description. Evidence from chromosome number, reciprocal crossing experiments and the absence of sesamolin from Sesernum latifolium all support our proposal that Sesarnum orien~ale var. malabaHcum Nat. is the progenitor of sesame. These facts suggest a closer relationship between the Indian relative (S. orientale vat. rnalabaricum) and cultivated taxa than between the East African taxon (S. latffolium) and the crop. Toxicological studies with sesamol [84, 85] reveal that a total of 20 proliferative lesions occurred in 134 rats fed sesamol. Sixteen of the lesions were benign, two were malignant and two were questionable. No such lesions were found in the controls, nor in rats receiving the two lowest dosages of sesamol. A report on toxicants occur-
dng naturally in foods [86] states that one component of insecticidal synergism appears to be the placing of extra demands on the animal's detoxification mechanism when they already have difficulty coping with the toxicant. The lignans in sesame oil are related to the hepatotoxin safrole [87] that increases the incidence of benign proliferative lesions in rats [86]. In view of what is now known about the biological activity of sesamin and sesamolin as insectical synergists and antioxidants, with an active methylenedioxyphenyl group, we question the use of sesame oil as a "control" in pharmacological experiments. It no longer seems appropriate to view sesame oil as "inert'. Experimental /so,t/onand ~ r o n - m t ~ ofo//componen~ sesame seeds werecrushedin a mortarandpestlewith CHCI=.Thefiltratewas passedthroughcottonwashedwith light petroleumto remove debris, and siiquotsof the filtrate applied directly onto TLC pines (ail~ ge~G). Triglyceride6 were rarnow~d by preparative TLC (silica gel G, light petroleum-Et~O-HOAc, 90:10:1). Plates were visualized with 2',7'-dichlorofluoroscein (0.1% in 50% EtOH). Lipids other than triglycerides were desorbed from silica gel with CHCI~ concentrated and rechromatographed (silica gel G-light pe~roleum-Et~O-HOAc, 70:30:1 or CHCla-benzene-MeOH,
30:20:0.5). Sampleswere flushedwith nitrogenand stored underrefrigerationafterthefinalconcen~ationstepto protectthe purified extractsfrom decomposition.All extractswere stored in the refrigerator. Isolationendpurifica~onof lignans,Sesaminand sasamolin were isolated from a preparativecolumn of silica gel. The column was packedin CHCI~.Sesameoil was placed on the column and eluted with light petroleum-E~O (95:5). The column was then washed with light petroleum-ErgO(9:1) to
TABLE 4. HERBARIUM SPECIMENS STUDIED Taxon
MO No.
Date
~e~rnum mngolense ~ ca4y~um $. capense £ ~ ~ n~/dum lap. ,'we~wa~ry~nurnlhl. and Said. Cer~to~eca z~oba C. ~#oba C, ~k~ba At#ub/e ~eccaB Ped~/~m murex Pre~ee ~ r ~ - u r n ~ eu~wn~;u$ $~arnol~mnu$ bu~eenus
2652241 2947307 2341123 2429666 2614251 2832733 2186743 244908? 1602340 2829866 2448044 2404153 2892717
20 August 1976 22 February 1979 1974 11 May 1976 2/~oril 1977 1 January 1979 22 Decern~er 1971 9 March 1977 1948 30 May 1976 14 April 1973 9 March 1975 November 1970
Countn/ M~lawi M~lawi Mozambique S.W. Africa South Africa Mozamt~que South Africa Swaziland Rhodesia Ghana Botew~n~ Soulh Africa Kenya
138
effect complete removal of triglyeerides. Lignans were removed with light petroleum:ErgO (50:50). Separation was monitored by TLC. Thi= work was ~upported in part by the University of Illinoi~ NSF Regional Instrumentation Facility, grant No. NSF CHE 79-16100. We thank the curator of the Missouri Botanical Garden herbarium for permk~ion to remove =eed material for this analysis.
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SESAMIN, SESAMOUNAND THE ORIGINOF SESAME
63. Waterman, P. G., Gray, A. L and Crichton, E. G. (1976) Biochem. Sys¢ Ecol. 4, 259. 64. Ishii, H., Ishikawa, T. and Haginiwa, J. (1977) YakugakuZasshi37, 890. 65. Deahpande, V. H. and Shastri, R. K. (1977) Ind. J. Chem. Sec~ B. 15, 95. 66. Stermitz, F. R., Caolo, M A. and Swinehart, J. A. (1980) PhytocherouS-,/15, 1469. 67. Weiss, S. G., Wa, M. T., Perdue, R. E. and Famsworth, N. R. (1975) J. Pharm. Sc~ 64, 95. 68. BhiravamurTy, P. V., Kanakale, RoD., Rao, E. V. and Sastw, K. V. (1979) Curt. Sc~ 48, 949. 68. Lavle, D., Levy, E. C., Cohen, A., Evenari, M. and Gutterman, Y. (1974) Nature, Lond. 249, 388. 70. Roy, S., Guha, R. and Chakrsborty, D. P. (1977) chem. Ind. 6. 231. 71. Sen, D. N. (1976) Ecophysiologica/Studies on Indian Weedsof Cultivated Fields with PanYcular Reference to bajre and ~7 .Cropsp. 119. US PL480 Report, University of Jodhpur, Jodhpur, India. 72. Atwal, A. S. and Manger, A. (1969) Ind. J. Entomol. 31, 286. 73. Varma, M. K., Sharma, H. C. and Pathak, V. N. (1978) P/ant Dis. Reporter62, 274. 74. Yu, L. S., Wilkinson, C. F. and Anders, M.W. (1980) Biochem. Pharrnac. 29, 1113.
139
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Sesamin, Sesamolin and the Origin of Sesame DOROTHEA BEDIGIAN*, DAVID S. SEIGLERI" and JACK R. HARLAN* *Crop Evolution Laboratory, Deparlment of Agronomy, University of Illinois, 1102S. Goodwin, Urbane, IL 61801, U.S~.; tDepartment of Plant Biology, University of Illinois, 505 S. Goodwin, Urbena, IL 61801, U.S~.
Kay Wind Index--Ses~rnurn/rid/cure; Pedaliaceae;lignan; phenylpropanoid; sesamin; =esamolln; TLC; NMR; evolution. Abslmct---Cultivars of sesame were screened to determine how widely lignens Occur. All lines tested contained sesamin and sesamolin. Sesamin and lesamolin in other species of Sesamumvaried. Some other genera in the Pedaliaceaealso possessed lignans. Phylochemical evidence as well as other data support the suggestion that the progenitor of sesame occurs in india.
Introduction The wild progenitor of sesame, Sesamum indicure L has not been established with certainty and the literature asserts with equal frequency that the center of origin of the crop is in Africa and Asia [1]. A recent proposal that Sesamum la~fo/ium Gillett is the progenitor of sesame and that the crop originated in Africa was accompanied by a division of the genus Sesamum into four sections [2]. The Pedaliaceae, to which sesame belongs, have been considered related most closely to the Bignoniaceae, Martyniaceae and Acanthaceae [3-5]. Although the use of chemical data to establish phylogenetic relationships is important [6-8], lignans have not been widely used for these analyses. Lignans are widespread in the plant kingdom [9, 10], occurring in heartwood, leaves and resinous exudates of plant families as distantly related as the Gingkoaceae and the Asteraceae. Approximately three dozen compounds are known at present [10]. Relationships of members of several families including the Asteraceae [11-13] have been studied with lignans but these compounds have not been used to study the phylogeny of plant families related to the Pedaliaceae. Seeds of sesame contain the lignans sesamin and sesamolin. These compounds are not found in other edible oils. The effects of strain, location grown, aging and frost damage on the sesamin, sesamolin and sesamol content of sesame seed (/bce/t~d~ &o~/lS84) 133
have been examined [14]. In a related species, Sesamum angolense Welw., the isolation and structure of a novel compound, sesangolin, an insecticidal synergist, has been reported [15]. The presence of sesamin was not reported. These workers obtained the oil from Pearman eta/. [16], who had submitted a seed sample to the director of the Royal Botanic Gardens, Kew, for identification; the seeds were believed to be those of S. ango/ense. Oil from the same seeds was reported to contain sesemin by Pearman eta/. [16]. The families considered to be the nearest relatives of the Pedaliaceae, the Bignoniaceae and the Acanthaceae, also contain lignans. Sesamin as well as a new lignan, peulownin ware isolated from the wood of Pau/ownia tomentosa Staud. (Bignoniaceae) [17, 18]; NMR spectral analysis of the stereochemistry of the latter compound shows structural similarity to sesamin and sesamoiin. Roots and stems of Phy//arthron comorense and Tabebuia rosea respectively (Bignoniaceae) yielded sesamin and peulownin [19]. The lignans sesamin, asarinin and sesamolin and a new compound, simplexoiin, were isolated from Just~'cia $imp/ex (Acanthaceae) [20]. Recent reports of sesamin in other related families include Hyp~'s tomentosa (Lamiaceae) [21], Aptos/mum spinescens Thunbg. (Scmphulariaceae) [22] and Gme/ina arborea Roxb. (Verbenaceae) [23, 24]. Other taxa from which sesamin and sesamolin have been reported are Acan~hopanax sentJcosus [25] and A. sess/liflorum (Araliaceae) [26]; Asarum sieboldii (Aristolochiaceae) [9, 27]; Anacyclus
134
pyrethrum (Asterceae) [28]; Artemisia absinthium group [12, 13, 29, 30]; Chrysanthemum cinerariaefo/ium (Asteraceae) [31]; C. frutescens [32]; C. indicum (Asteraceae) [33]; Diotis rnari~'ma (Asteraceae) [34]; Ageratina, Critoniaand Fleischmannia (Asteraceae), formerly part of the genus Eupatorium, [35]; Otanthus maritimus (Asteraceae) [36, 37]; Alnus g/u#'nosa (Betulaceee) [38]; Salicomia europaea (Chenopodiaceee) [39]; Austrocedrus chi/ensis (Cupressaceee) [40]; CharnaecypaHs (Cupressaceee) (a garden species that has hibalactone which upon reduction yields hinokinic acid and d-sesamin) [41]; Gingko biloba (Gingkoaceae) [42, 43]; Machi/usglaucescens(Lauraceee) [44, 45]; Ocotea usarnbarensis (Lauraceee) [46]; species of Magno/ia (Magnoliaceae) [47-49]; Talauma hodgsonii (Magnoliaceae) [50]; Picea abies (Pinaceee) [38]; Macropiper exce/surn (Piperaceee) [51]; the genus Piper (Piperaceee) [52-58]; Evodia micrococca var. pubescens (Rutaceae) [27]; Fagaraxanthoxyloides(Rutaceae) [59]; F/indersia pubescens (Rutaceae) [60]; and the genus Zanthoxylum (Rutaceee) [61-66]. Lignans are active constituents of certain medicinal plants [10]. Podophyllin, a complex resinous extract of may apple, Podophyllum peltaturn, has been used as a powerful cathartic. The principal lignan constituent, podophyllotoxin, is of interest as it has cytotoxic action similar to colchicineo Podophyllotoxin and other lignans that possess the partially reduced naphthalene nucleus have shown some promise in treating certain types of neoplasms [10, 67]. However, when constituents of Hyptis tomentosa (Lamiaceae) were screened for anticancer agents, sesamin was found to be inactive [21]. It has been reported that sesamin and sesamolin may play a role in natural seed dormancy. Germination inhibition by sesamin and sesamolin is considerably greater with peanut and cucumber seeds than with rice, where only retarded coleoptile growth occurred [68]. The effect is more pronounced on lipid-storing seeds than on seeds containing carbohydrate as storage product, and it has been suggested that the effect is on the end product initiating enzymes controlling lipid mobilization [68]. A germination inhibitor isolated from Aegilops ova~a [69] bears a close structural resemblance to sesame lignans. Leached hulls inhibited the germination of Lactuca achenes. Inhibition was
DOROTHEABEDIGIAN,DAVIDS. SEIGLERAND JACK R. HARLAN
considerably stronger in light than in darkness. The authors claimed that it was a lactone. This report was questioned [24] and the compound later shown to be the lignan acanthotoxin [70]. This compound is also found in Zanthoxylum acanthopodium (Rutaceae) and has spectral properties identical with those of justicidin E, isolated from Justicia procurnbens (Acanthaceae) [70]. Extracts of wild sesame seed from India inhibited seedling growth of cultivated sesame [71]. Root exudates of sesame are repellant to root knot nematodes of eggplant, tomato, potato and okra [72, 73]. Sesame was as potent a nematicide as marigold. Field trials showed that intercropping with sesame is economically beneficial as the nematode problem is eliminated and in addition the sesame can be harvested. The action of sesamin and sesamolin as inhibitom of mixed function oxidases [74] is due to the methylenedioxyphenyl group. This group has been recognized previously as an effective inhibitor of microsomal oxidation. Sesamin and sesamolin have been used for practical applications as antioxidants and insecticides. In the course of testing pyrethrum extracts in combination with a number of vegetable and fish oils, it was found that sesame oil, but not others, markedly increased the effectiveness of pyrethrum insecticides [75]. Sesamin and sesamolin enable sesame oil to resist oxidative rancidity [76-78], an action that is also attributed to the methylenedioxyphenyl group. Sesamin and sesamolin are used as active ingredients in antioxidants, antiseptics, bactericides, viricides, disinfectants, moth repellants and anti-tubercular agents. These uses are mentioned in a series of patents. Sesame oil has been used as a control in pharmacological experiments. We undertook this study to learn more about the phylogenetic relationships of sesame and the Pedaliaceae. We were seeking new data to help ascertain the origin of the cultivated crop and to learn the extent to which sesemin and sesamolin occur in related taxa. Neighboring species, genera and families were examined. We were also interested in a practical procedure for the determination of sesamin and sesemolin by TLC to screen seed oils of members of this group. We attempted to develop an analytical method to
135
SESAMIN, SESAMOLIN AND THE OffiGIN OF SESAME
screen oils rapidly as well as to isolate and identify sesamin and sesemolin with NMR spectropscopy in order to determine structure and to assess purity. Results Oils of the taxa studied were separated on silica gel by TLC or column chromatography. Sesamin and sesamolin ware resolved from other seed oil compounds. We also defined a solvent system for TLC that would separate the lignans of sesame. After removing triglycerides, the best separation was obtained with a chloroform, benzene and MeOH mixture (60:40:1). Sesamolin travelled nearest the solvent front (Rf 0.54) and sesemin lagged behind it (Rf0.36). Up to six other spots ware observed under u.v. light after spraying the plates with 2',7'-dichlorofiuorescein, or after acid charring, but these ware found to be non-lignan lipids by NMR spectroscopy. The first step in the taxonomic comparisons was to examine 50 cultivars of sesame that originated in different geographic regions of the world. We found no significant differences in sesamin and sesemolin content among the cultivers. All samples tested possessed both lignans. As no lines of sesame contained sesamol, a hydrolysis product [77] of sesamolin, it did not appear that our procedure was accompanied by hydrolytic degradation. We attempted to locate where in the seed the lignans are stored. Dissection of seeds to remove the oily endosperm that wraps around the embryo proved to be difficult and contamination of the embryo fraction with some endosperm tissue was unavoidable. Our results show that the embryo fraction lacks sesemolin, which is associated only with endosperm tissue. Sesamin was found in both portions. We also examined near relatives of cultivated sesame to see how widespread these lignans are in the genus Sesemum (Table 1). Sesamin and sesemolin were found in seed oils of S. angolense, S. angustJfolium, S. calycinum, S. indicum, S. offentale var. malabaricum and a weedy escape from Australia. Oils of $. laUfoliurn and S. radiatum contained sesemin but not sesamolin. The presence of lignans in other species was more difficult to establish. These compounds were absent from fresh seed of Sesarnum alatum. Even when relatively large amounts of seed were
TABLE 1. UGNAN CONTENT OF SEEDS OF SPECIES OF SESAMUM Species
Seed
S~samin
Selamolin
$~s~mum a/a~urn Thonn. & engo/en~e Welw. ~ a n ~ m ( Q ~ . ) E~I. ~ ~um Welw. ~ ~ Bu~.
f h f h h
-+ + + -
-+ + + T~ce
~ ~um
L.
f
+
+
~ ~ ~ ~
Gi~ ~r. ~ ~ . ~um.
f f h f
+ + T~e +
-+ -
h h
---
---
f
+
+
~ m ~ ~ ~
~r. ex. H ~ m ~ ~ .
S~m~.~. ~ m Ihl. ~ ~ i . ~ ~ m We~. ex. ~ . U. ~ I. No. ~ : ~ ~ ~ ~lia.
Seed source code: f--fresh seed; h-herbarium specimen. Voucher specimen numbem given in Experimer,~al section.
extracted as described below, the sample contained only non-lignan lipid$ as indicated by NMR spectral analysis. TLC also confirmed that lignans were absent. In this study, seeds of Sesemum angolense from herbarium specimens showed the presence of both sesamin and sesamolin. A spot identical in Rf to sesamin was observed; we cannot exclude the possibility, however, that the compound is sesangolin, not sesamin. Confirmation with fresh seed material and NMR spectroscopy could resolve this problem. The use of TLC of lignans as a diagnostic tool can be illustrated by the following example. The germplasm collection maintained by the Agricultural Research Corporation, Kadugli, Sudan, contains line 144 that was identified as Sesamum indicum. The shape and color of the corolla, and overall plant habit, and the fact that this cultivar remained green well after all other lines in the nursery had begun to senesce suggested that it differed greatly from other cultivars of sesame. It was the last line to be harvested. Testa ornamentation on seeds from line 144 appeared similar to those of specimens of Sesarnurn radiaturn (D. Bedigian, unpublished data). Extracts of both samples were compared by TLC. The chromatograms were identical and it is probable that line 144 is actually S. radiatum. Other genera of the family Pedaliaceae ware examined to determine the extent to which they
136
DOROTHEA BEDtGIAN, DAVID S. SEIGLER AND JACK R. HARLAN
TABLE 2, UGNAN CONTENT OF SEEDS OF RELATED GENERA OF SESAMUM Species Ceratotheca sesa~ide$ Endl. Cemmff~va 1~7obaMeyer ex Bemh. Ho/ub~ s~cca= Oily. Pedalium murex L. P~t~e z~nguebericum Gay Ptetodiscus aur~n~cus Welw. P.o~en~~termphy//~ Gay ex Del. ~ff~mnus ~lse~nu$ Engl.
Seed
Sesamin
Sesamolin
f h h h h h f h
÷ Trace Trace Trace Trace ÷
Trace -----+
contain lignans (Table 2). These genera vary considerably in their content of lignans. The presence of lignans in plant families related to the Pedaliacaae was also examined. Fresh seeds of the taxa listed (Table 3) were studied (nomenclature according to Hortus Third) [79]. Sesemin and sesemolin were not found in the seed oils of representatives of other plant families that we tested. Several of these did contain a similar sedes of non-lignan lipids, but NMR studies showed that those compounds were not lignans. Although traces of other lignans could be present, they are not major components as they are in sesame. The wild relatives of sesame, including S. latifol/um, have dormant seeds; this was confirmed by Sudanese agronomists (M. O. Khidir and M. A. Mahmoud, pers. comm.). Dormancy of both S. latifoliurn and S. orientMe var. malabaricum was broken only by leaching the seeds overnight in running tap water, followed by 3% hydrogen peroxide soaks. Germination was still only 20% (D. Bedigian, unpublished date). TABLE 3. TAXA STUDIED IN PLANT FAMILIES RELATED TO THE PEDALIACEAE
Acanthus rnollis L. (Acanthaceae) Ca=/pa b/geoe/o/de~Walt. (Bignoni~ceae) Peu/oww/e mmento=a ('rhunb.) St~ud. (Bidnoniaceae) Bor~go o f f l ~ / ~ L. (Boraginaceae) Omphalo#es I~ifoli~ (L.) Moench (Borlginaceae) Coleus x hyt~du= VOLS. (Lamlaceae) S~h,~ ver//cB~= L. vat. ~tba (Lamiaceae) Pmbosc/de~ ~ (MB.) TheN. (Mortynioceae) from Arizoea ~ ~ (Mill.! The¢. ~ l from O~dahoma Digi~li$ ,ou,-pureaL. (S~-.7#~d~riaceae) Mk~u/us ~ Dougl. ex Benth. ( S c ~ u l l r i a c e a e ) PenBtemon c.~ml~nubtus (C~v.) Willd, (Scrol~hulari=ceae) Torenia vio~cea (Azaola) Penn. (Scrophularlaceae) C.len~endrum ~ Dode ~ = e ) /.~nCanac~m,~r#L. Nedoenaceoe)
Discussion We have been able to confirm the identity of two lignans, sesamin and sesamolin in sesame oil. These are recognizable under UV light on TLC plates, but their identification was also established by NMR spectroscopy. By the same methods, we have shown that S. alarum consistently lacks these constituents. Sesangolin does not appear to be present in S. ango/ense. Although we cannot exclude the presence of other lignans, we saw no evidence for the presence of this compound, as the lignans of S. angolense had the same Rt values as those of sesame. As the materials previously studied were apparently not supported by voucher specimens (none is cited), we cannot be sure of the identification of the plant examined. Additionally this report appeared prior to the description of S. latifolium [80], a species with which S. angolense might easily be confused and this makes the identification even more tentative. The absence of lignans from both S. alarum and S. capense is noteworthy as both are species with winged seeds in the section Sesamoptaris, that is relatively isolated within the family [2]. Annotations on herbarium specimens show that there is disagreement concerning the identification and synonomy of these two species (D. Bedigian, unpublished data). Although sesamin and sesamolin are reported to occur in members of related families, investigation of a series of plants from these families, chosen at random, did not reveal sesamin and sesamolin in families other than the Pedaliaceae. As mentioned previously, Sesamum latifolium has been suggested as the ancestor of sesame [2]. This species occurs in East Africa and contrary .to previous reports, is widely distributed throughout the Sudan [81]. The chromosome number of S. latifolium is 2n-32 [2]. Reciprocal crosses between the crop and S. latifolium have been attempted extensively by Sudanese breeders (M. O. Khidir, pers. comm.; D. Bedigian, unpublished data), but have never succeeded. Only a few shrunken seeds produced in a few tiny twisted capsules were obtained. Attempts to germinate these seeds were futile (D. Bedigian, unpublished data). Samples of seeds of S. /atJfolium consistently lack sesamolin. We regard the nearest wild relative of cultivated sesame as the taxon known in India as the
SESAMIN, SESAMOLIN AND THE ORIGINOF SESAME
137
wild gingelly of Malabar, named Sesamum orientale var. malabaHcum Nar. in the original description [82]. This taxon is the probable ancestor of the crop and like cultivated sesame, contains both sesamin and sesamolin. Numerical analyses of the patterns of morphological variation in sesame [83] confirm the nearness of relationship between this wild Indian variety and the crop. The closeness of the relationship between the crop and its putative ancestor is also shown by reciprocal crosses, which were fully ferlJle using ver. rnalabaHcurn as both male and female parent. The chromosome number of S. orien~ale vat. malabaHcum is 2n-26, the same as the crop [82]. As no Latin description was given atthe time of publication [82], the report provides information about the plant but does not constitute a valid taxonomic description. Evidence from chromosome number, reciprocal crossing experiments and the absence of sesamolin from Sesernum latifolium all support our proposal that Sesarnum orien~ale var. malabaHcum Nat. is the progenitor of sesame. These facts suggest a closer relationship between the Indian relative (S. orientale vat. rnalabaricum) and cultivated taxa than between the East African taxon (S. latffolium) and the crop. Toxicological studies with sesamol [84, 85] reveal that a total of 20 proliferative lesions occurred in 134 rats fed sesamol. Sixteen of the lesions were benign, two were malignant and two were questionable. No such lesions were found in the controls, nor in rats receiving the two lowest dosages of sesamol. A report on toxicants occur-
dng naturally in foods [86] states that one component of insecticidal synergism appears to be the placing of extra demands on the animal's detoxification mechanism when they already have difficulty coping with the toxicant. The lignans in sesame oil are related to the hepatotoxin safrole [87] that increases the incidence of benign proliferative lesions in rats [86]. In view of what is now known about the biological activity of sesamin and sesamolin as insectical synergists and antioxidants, with an active methylenedioxyphenyl group, we question the use of sesame oil as a "control" in pharmacological experiments. It no longer seems appropriate to view sesame oil as "inert'. Experimental /so,t/onand ~ r o n - m t ~ ofo//componen~ sesame seeds werecrushedin a mortarandpestlewith CHCI=.Thefiltratewas passedthroughcottonwashedwith light petroleumto remove debris, and siiquotsof the filtrate applied directly onto TLC pines (ail~ ge~G). Triglyceride6 were rarnow~d by preparative TLC (silica gel G, light petroleum-Et~O-HOAc, 90:10:1). Plates were visualized with 2',7'-dichlorofluoroscein (0.1% in 50% EtOH). Lipids other than triglycerides were desorbed from silica gel with CHCI~ concentrated and rechromatographed (silica gel G-light pe~roleum-Et~O-HOAc, 70:30:1 or CHCla-benzene-MeOH,
30:20:0.5). Sampleswere flushedwith nitrogenand stored underrefrigerationafterthefinalconcen~ationstepto protectthe purified extractsfrom decomposition.All extractswere stored in the refrigerator. Isolationendpurifica~onof lignans,Sesaminand sasamolin were isolated from a preparativecolumn of silica gel. The column was packedin CHCI~.Sesameoil was placed on the column and eluted with light petroleum-E~O (95:5). The column was then washed with light petroleum-ErgO(9:1) to
TABLE 4. HERBARIUM SPECIMENS STUDIED Taxon
MO No.
Date
~e~rnum mngolense ~ ca4y~um $. capense £ ~ ~ n~/dum lap. ,'we~wa~ry~nurnlhl. and Said. Cer~to~eca z~oba C. ~#oba C, ~k~ba At#ub/e ~eccaB Ped~/~m murex Pre~ee ~ r ~ - u r n ~ eu~wn~;u$ $~arnol~mnu$ bu~eenus
2652241 2947307 2341123 2429666 2614251 2832733 2186743 244908? 1602340 2829866 2448044 2404153 2892717
20 August 1976 22 February 1979 1974 11 May 1976 2/~oril 1977 1 January 1979 22 Decern~er 1971 9 March 1977 1948 30 May 1976 14 April 1973 9 March 1975 November 1970
Countn/ M~lawi M~lawi Mozambique S.W. Africa South Africa Mozamt~que South Africa Swaziland Rhodesia Ghana Botew~n~ Soulh Africa Kenya
138
effect complete removal of triglyeerides. Lignans were removed with light petroleum:ErgO (50:50). Separation was monitored by TLC. Thi= work was ~upported in part by the University of Illinoi~ NSF Regional Instrumentation Facility, grant No. NSF CHE 79-16100. We thank the curator of the Missouri Botanical Garden herbarium for permk~ion to remove =eed material for this analysis.
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