Patent Application: US-73875407-A

Abstract:
a method for detecting herbs or plants of the aristolochia genus in herbal products and botanicals may be based on the sequence information in the trnl - trnf region and / or the psba - trnh region . the trnl - trnf region and / or the psba - trnh of the chloroplast dna contain a number of segments which are highly characteristic of the plants of the aristolochia genus . by this method , the degree of similarity in one or more of these segments between an herbal or plant material of unknown identification and reference dna sequence of a known material can be used to authenticate the unknown material .

Description:
reference will now be made in detail to a number of particular embodiments of the invention as examples to facilitate the understanding of the present invention . exemplary embodiments of the invention are described in detail , although it will be apparent to those skilled in the relevant art that some features that are not particularly important to an understanding of the invention may not be shown for the sake of clarity . on the other hand , details provided in connection with the particular embodiment are by example only , of which various changes and modifications may be effected by one skilled in the art without departing from the spirit or scope of the invention . according to the pharmacopoeia of the people &# 39 ; s republic of china ( the 2005 edition ), the chinese herb muxiang ( or in chinese ) is derived from aucklandia lappa and some species of the daisy family ( compositae ). it is sometimes adulterated by aristolochia debilis , which is better known as qingmuxiang ( or in chinese ) and contains the carcinogenic aristolochic acids . as shown in fig1 and 2 , the trnl - trnf ( fig1 a and 1 b ) and psba - trnh ( fig2 a and 2 b ) sequences can be used to differentiate the genuine and adulterate species of muxiang . by aligning the aristolochia trnl - trnf sequences with those of genuine muxiang species ( fig1 a and 1 b ), it is clear that there are many differences ; the more prominent ones are shown below : ( i ) ‘ ttaaaa ’ in aristolochia species but ‘ ttmtaaaa ’ in genuine muxiang species ( bases 93 - 101 in fig1 a ), referred to as genetic marker 1 ; ( ii ) ‘ gtactgaaata ’ in aristolochia species but ‘ atmta ’ in genuine muxiang species ( bases 367 - 377 in fig1 a ), referred to as genetic marker 2 . similarly , aligning the aristolochia psba - trnh sequences with those of the genuine muxiang species ( fig2 a and 2 b ), shows that there are many differences ; the more prominent ones are shown below : ( i ) ‘ gmgga ’ in aristolochia species but ‘ gmctaaaaaagga ’ in genuine muxiang species ( bases 106 - 119 in fig2 a ), referred to as genetic marker 3 ; ( ii ) ‘ cagg ’ in aristolochia species but ‘ ctaaaatagataaaaatta taattttgattatttattgcttttatttcagaaataagaaaga mtaatatgctcttttttttatgttmtggaaaaataaaatat agtaatagtagataatactagatmtagg ’ in genuine muxiang species ( bases 335 - 468 in fig2 a ), referred to as genetic marker 4 . according to the pharmacopoeia of the people &# 39 ; s republic of china ( the 2005 edition ), mutong ( or in chinese ) is derived from akebia quinata , akebia trifoliata , akebia trifoliata var . australis and may also include chuanmutong ( or in chinese ), which is derived from clematis armandii and clematis montana . mutong is sometimes adulterated by guanmutong ( aristolochia manshuriensis ), known as in chinese , which contains carcinogenic aristolochic acids . as shown in fig3 and 4 , the trnl - trnf ( fig3 a and 3 b ) and psba - trnh ( fig4 a and 4 b ) sequences can be used to differentiate the genuine and adulterate species of mutong . although the sequences of trnl - trnf region of akebia quinata ( genbank ay651844 ; af335297 ), akebia trifoliata , ( genbank af335294 ) and aristolochia manshuriensis ( genbank ay689184 ), respectively , have been published for phylogenetic studies by three independent research groups ( quandt et al ., 2004 ; wang et al ., 2002 ; neinhuis et al ., 2005 ), they have never been considered for any authentication purposes . by aligning the aristolochia trnl - trnf sequences with those of genuine mutong species ( fig3 a and 3 b ), the differences are clear . the more prominent ones are shown below : ( i ) ‘ agagtggga ’ in aristolochia species but ‘ agaaagga ’ in genuine mutong species ( bases 299 - 307 in fig3 a ), referred to as genetic marker 5 ; ( ii ) ‘ gactatca ’ in aristolochia species but ‘ agtca ’ in genuine mutong species ( bases 498 - 505 in fig3 a ), referred to as genetic marker 6 . similarly , by aligning the aristolochia psba - trnh sequences with those of the genuine mutong species ( fig4 a and 4 b ), it can be noted that among many differences , the more prominent ones are shown below : ( i ) absent in aristolochia species but ‘ gaaac ’ in genuine mutong species ( bases 421 - 425 in fig4 a ), referred to as genetic marker 7 ; ( ii ) absent in aristolochia species but ‘ tttgag ’ in genuine mutong species ( bases 608 - 613 in fig4 a ), referred to as genetic marker 8 . according to the pharmacopoeia of the people &# 39 ; s republic of china ( the 2005 edition ), fangji ( or in chinese ) is derived from stephania tetrandra . it is sometimes adulterated by guangfangji ( aristolochia fangchi and related aristolochia species ) referred to as in chinese , which contains carcinogenic aristolochic acids . it can also be confused with mufangji ( or ” in chinese ) derived from cocculus trilobus and related cocculus species . as detailed in fig5 and 6 , the trnl - trnf ( fig5 a and 5 b ) and psba - trnh ( fig6 a and 6 b ) sequences can be used to differentiate aristolochia species from other plant species used as fangji . by aligning the aristolochia trnl - trnf sequences with those of genuine fangji species ( fig5 a and 5 b ), it is clear that there are many differences ; the more prominent ones are listed below : ( i ) ‘ gtggga ’ in aristolochia species but ‘ aaagga ’ in genuine fangji species ( bases 298 - 303 in fig5 a ), referred to as genetic marker 9 ; ( ii ) ‘ atcgactatca ’ in aristolochia species but ‘ atcagtca ’ in genuine fangji species ( bases 479 - 489 in fig5 a ), referred to as genetic marker 10 ; ( iii ) ‘ tttttcacttact ’ in aristolochia species but ‘ tttctcgttca ct ’ in genuine fangji species ( bases 764 - 776 in fig5 a ), referred to as genetic marker 11 ; ( iv ) ‘ tccatatatgg ’ in aristolochia species but ‘ tct ’ in genuine fangji species ( bases 868 - 878 in fig5 a ), referred to as genetic marker 12 ; ( v ) ‘ att ’ in aristolochia species but ‘ attccaagg ’ in genuine fangji species ( bases 981 - 988 in fig5 a ), referred to as genetic marker 13 ; ( vi ) ‘ tagtagga ’ in aristolochia species but ‘ tatgggga ’ in genuine fangji species ( bases 1096 - 1103 in fig5 a ), referred to as genetic marker 14 . similarly , by aligning the aristolochia psba - trnh sequences with those of the genuine fangji species ( fig6 a and 6 b ), it becomes clear that there are many differences ; the more prominent ones are listed below : ( i ) ‘ cgt ’ in aristolochia species but ‘ cgttgaa ’ in genuine fangji species ( bases 102 - 108 in fig6 a ), referred to as genetic marker 15 ; 1 ( ii ) absent in aristolochia species but ‘ gtttcccgacatc ’ in genuine fangji species ( bases 385 - 397 in fig6 a ), referred to as genetic marker 16 . according to the pharmacopoeia of the people &# 39 ; s republic of china ( 1977 edition ), baiying ( or in chinese ) is derived from solanum lyratum . it is sometimes adulterated by xungufeng ( aristolochia mollissima ), known as in chinese , which contains carcinogenic aristolochic acids . as detailed in fig7 and 8 , the trnl - trnf ( fig7 a and 7 b ) and psba - trnh ( fig8 a and 8 b ) sequences can be used to differentiate the genuine from the adulterate species of baiying . aligning the aristolochia trnl - trnf sequences with those of genuine baiying species ( fig7 a and 7 b ), reveals that there are many differences ; the more prominent ones are listed below : ( i ) ‘ taaaaa ’ in aristolochia species but ‘ taacaaaaa ’ in genuine baiying species ( bases 94 - 102 in fig7 a ), referred to as genetic marker 17 ; ( ii ) ‘ gaaata ’ in aristolochia species but ‘ gaatactata ’ in genuine baiying species ( bases 370 - 379 in fig7 a ), referred to as genetic marker 18 ; ( iii ) ‘ actgtg ’ in aristolochia species but ‘ acaagcctttatg ’ in genuine baiying species ( bases 827 - 839 in fig7 a ), referred to as genetic marker 19 ; ( iv ) ‘ tttatttaggtaagtta ’ in aristolochia species but ‘ tttgagaaaatttttta ’ in genuine baiying species ( bases 991 - 1007 in fig7 a ), referred to as genetic marker 20 ; ( v ) ‘ gagaat ’ bsent in aristolochia species but ‘ gagattggaat ’ in genuine baiying species ( bases 1108 - 1119 in fig7 a ), referred to as genetic marker 21 . similarly , by aligning the aristolochia psba - trnh sequences with those of the genuine baiying derived from solanum species ( fig8 a and 8 b ), it is clear that there are many differences ; the more prominent ones are listed below : ( i ) ‘ gaagga ’ bsent in aristolochia species but ‘ gaaaagaaagg a ’ in genuine baiying species ( bases 106 - 117 in fig8 a ), referred to as genetic marker 22 ; ( ii ) absent in aristolochia species but ‘ tgttcctcttgttgctaat gttactatatcttttttatttcatttcacaaaaaatataatttt tacttcatattcttatctttgaaataataa ’ in genuine baiying species ( bases 327 - 419 in fig8 a ), referred to as genetic marker 23 ; ( iii ) absent in aristolochia species but ‘ aaataataatatcattgaa ataagaaagaagagaaatattcgaacttta ’ in genuine baiying species ( bases 434 - 483 in fig8 a ), referred to as genetic marker 24 . according to the pharmacopoeia of the people &# 39 ; s republic of china ( the 2005 edition ), madouling ( or in chinese ) is derived from aristolochia contorta and aristolochia debilis , which contain carcinogenic aristolochic acids and thus must be used more carefully . its alternatives include dabaihe , which is known as in chinese and is derived from cardiocrinum giganteum and related liliaceae plants . the trnl - trnf ( fig9 a and 9 b ) and psba - trnh ( fig1 a and 10 b ) sequences can used to differentiate the genuine and alternative species of madouling . by aligning the aristolochia trnl - trnf sequences with those of dabaihe species ( fig9 a and 9 b ), it is clear that there are many differences ; the more prominent ones are listed below : ( i ) ‘ tttcacaaa ’ in aristolochia species but ‘ tttagtctc ’ in dabaihe species ( bases 807 - 815 in fig9 a ), referred to as genetic marker 25 ; ( ii ) ‘ tacgtacaaatgcccatccatatatgg ’ in aristolochia species but absent in dabaihe species ( bases 865 - 891 in fig9 a ), referred to as genetic marker 26 . similarly , by aligning the aristolochia psba - trnh sequences with those of dabaihe species ( fig1 a and 10 b ), it becomes clear that there are many differences ; the more prominent ones are listed below : ( i ) ‘ cg ’ in aristolochia species but ‘ cattct ’ in dabaihe species ( bases 82 - 87 in fig1 a ), referred to as genetic marker 27 ; ( ii ) ‘ tacccaa ’ in aristolochia species but ‘ tacctttgtatcttg ctaaagatattgctccctttttttggccaa ’ in dabaihe species ( bases 120 - 164 in fig1 a ), referred to as genetic marker 28 ; ( iii ) ‘ aagg ’ in aristolochia species but ‘ aagctaaaatctttt agctaga ’ in dabaihe species ( bases 412 - 433 in fig1 a ), referred to as genetic marker 29 . according to guanmutongshenduxingyanjiu chinese ) zhushalian ( or in chinese ) is derived from aristolochia kaempferi and related species . its alternatives include roots derived from dioscorea cirrhosa , known as shuliang ( or in chinese ), and related species in the family dioscoreaceae . the trnl - trnf ( fig1 a and 11 b ) and psba - trnh ( fig1 a and 12 b ) sequences can used to differentiate the genuine and alternative species of zhushalian . by aligning the aristolochia trnl - trnf sequences with those of shuliang species ( fig1 a and 11 b ), it is clear that there are many differences ; the more prominent ones are listed below : ( iii ) ‘ atcctgtt ’ in aristolochia species but ‘ atctttatttgtt ttgtt ’ in shuliang species ( bases 118 - 135 in fig1 a ), referred to as genetic marker 30 ; ( iv ) ‘ tcagaagcaga ’ in aristolochia species but ‘ tcaaccga agttga ’ in shuliang species ( bases 471 - 484 in fig1 a ), referred to as genetic marker 31 ; ( v ) ‘ atcgactatcacaccaga ’ in aristolochia species but ‘ atcattgattctaga ’ in shuliang species ( bases 512 - 529 in fig1 a ), referred to as genetic marker 32 ; ( vi ) ‘ tgcatcgagaatggtcgg ’ in aristolochia species but ‘ tgcgagaaaagaaaatggttggg ’ in shuliang species ( bases 1074 - 1098 in fig1 a ), referred to as genetic marker 33 . similarly , by aligning the aristolochia psba - trnh sequences with those of shuliang ( fig1 a and 12 b ), it is clear that there are many differences ; the more prominent ones are listed below : ( iv ) ‘ acctggc ’ in aristolochia species but ‘ acttagc ’ in shuliang species ( bases 38 - 44 in fig1 a ), referred to as genetic marker 34 ; ( v ) ‘ atacccaat ’ in aristolochia species but ‘ atatcaata ’ in shuliang species ( bases 115 - 123 in fig1 a ), referred to as genetic marker 35 . when the sequences of all the herbs in the above five examples are aligned together , it is noted that , in general , the aristolochia trnl - trnf region has specific sequence segments which are characteristic of the plants of the aristolochia genus when compared with the other plant species of muxiang , mutong , fangji , baiying and dabaihe ( fig1 ). some examples of the characteristic segments are as follows : ( i ) ‘ agtgggatg ’ in aristolochia species ( bases 313 - 321 in fig1 ), referred to as genetic marker 36 ; ( ii ) ‘ aatttcagaag ’ in aristolochia species ( bases 472 - 482 in fig1 ), referred to as genetic marker 37 ; ( iii ) ‘ actatcacacc ’ in aristolochia species ( bases 525 - 535 fig1 ), referred to as genetic marker 38 . similarly , the aristolochia psba - trnh region also has some specific segments which are characteristic of plants of the aristolochia genus compared with the other plant species muxiang , mutong , fangji , baiying and dabaihe ( fig1 ). an example is : ( i ) ‘ ctcaattcacta ’ in aristolochia species ( bases 172 - 183 in fig1 ), referred to as genetic marker 39 . in the above , each genetic marker ( i . e ., of genetic markers 1 - 39 ) comprises a number of sequences , each for a particular species as defined and aligned together in the corresponding figs . the sequences of each genetic marker are of the same or different length , also detailed in the corresponding figs . the method of the present invention as exemplified in the foregoing examples can be utilized in many circumstances to determine whether aristolochia herbs are present in botanicals or herbal products by making an extract of the total dna from the samples of concern , followed by amplification of either or both of the trnl - trnf and psba - trnh regions by polymerase chain reaction ( pcr ) and then by dna sequencing . by comparing and matching the resulting trnl - trnf and psba - trnh sequences to those of the aristolochia herbs , the matched samples would belong to aristolochia . as demonstrated in the foregoing examples , there are a number of sequence segments within the trnl - trnf and psba - trnh regions that are characteristic of the plants of the aristolochia genus . for particular cases , it may not be necessary to amplify and sequence the entire trnl - trnf region and / or the entire psba - trnh region , but amplifying and sequencing a portion of the trnl - trnf region and / or the psba - trnh region may be sufficient for a particular purpose in a particular situation . for pcr and dna sequencing of the trnl - trnf region , the forward primer is tab c ( 5 ′- cgaaatcggtagacgctacg - 3 ′), and the reverse primer is tab f ( 5 ′- atttgaactggtgacacgag - 3 ′). for pcr and dna sequencing of psba - trnh region , the forward primer is ( 5 ′- ggtatgcatgmcgtmtgctc - 3 ′), and the reverse primer is ( 5 ′- cgcgcatggtggattcacaaatc - 3 ′). while there have been described and pointed out fundamental novel features of the invention as applied to preferred embodiments thereof , it will be understood that various omissions and substitutions and changes , in the form and details of the processes and methods illustrated , may be made by those skilled in the art without departing from the spirit of the invention . for example , it is expressly intended that all combinations of those elements of method steps which perform substantially the same function in substantially the same way to achieve the same results are within the scope of the invention . the invention is not limited by the embodiments described above which are presented as examples only but can be modified in various ways within the scope of protection defined by the appended patent claims . arlt , v . m . ; 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