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Clonal Propagation of Cephaelis ipecacuanha (II): Characteristics of Regenerated Plants Field-Cultivated in two Districts
J Plant Physiol. Vol. 144. pp. 22-25 (1994)
Clonal Propagation of Cephaelis ipecacuanha (II): Characteristics of Regenerated Plants Field-Cultivated in two Districts KAyO
Y OSHIMATSU,
KAZUYA AOI*,
and KOICHIRO
SHIM OMURA
Tsukuba Medicinal Plant Research Station, National Institute of Health Sciences, 1 Hachimandai, Tsukuba, Ibaraki, 305 Japan
* Tanegashima Experiment Station of Medicinal Plants, National Institute of Health Sciences, 17019 Noma, NakatanecM, Kumage-gun, Kagoshima, 891-36 Japan
Received March 24, 1993 . Accepted December 10, 1993
Summary Ipecac plants, Cephaelis ipecacuanha A. Richard, were c10nally propagated through shoot-tip culture and field-cultivated at Tsukuba (Lat. 36° N, Long. 140° E) and Tanegashima (Lat. 30° 30' N, Long. 131 °E) in Japan. Growth and alkaloid content were periodically measured, and influence of environmental conditions was investigated. In Tanegashima, plants grew gradually from June to October, showing homogeneous growth of aerial parts. However the aerial parts were significantly damaged between January and February when minimum average monthly temperature was below 10°C. In Tsukuba, the plants did not grow well, but the aerial parts were alive until October. The alkaloid contents in the roots increased from June to December in Tanegashima, while in Tsukuba they decreased once in September and October. The maximum alkaloid content in the roots was observed in December, both in Tsukuba (2.4 % DM cephaeline, 1.0 % DM emetine) and Tanegashima (3.2 % DM cephaeline, 1.8 % DM emetine).
Key words: Cephaelis ipecacuanha, Rubiaceae, alkaloids, emetine, cephaeline, field-cultivation, regenerated plant, shoot-tip culture.
Abbreviations: HPLC = high performance liquid chromatography; FM = fresh mass; DM = dry mass; C.V. = coefficient of variation.
Introduction
Cephaelis ipecacuanha A. Richard (Family: Rubiaceae), native to the Amazon region and rich in alkaloids, is one of the important medicinal plants. Its dried roots and rhizomes, called ipecac, are commonly used as an expectorant in bronchitis, croup, asthma, and whooping cough, as an emetic in the cases of poisoning, and an amoebacide in amoebic dysentery (Trease and Evans, 1989). The supply of ipecac has mainly relied on collection of wild plants because only modest success of commercial cultivation has been achieved in Malaya and India (Trease and Evans, 1989). The best quality ipecac, Rio drug, is now be© 1994 by Gustav Fischer Verlag, Stuttgart
coming difficult to obtain because tropical forests are being gradually destroyed. Plant tissue culture has been considered as a promising tool for preservation of plant resources as well as the commercial mass propagation of plants (Rao and Lee, 1982). Recently we established micropropagation of ipecac plants through shoot-tip culture (Ikeda et al., 1988). However, for the tissue culture method to replace conventional methods, it is of importance to investigate the characteristics of growth and alkaloid content of the regenerated plants that are cultivated in the field. In this paper, we describe the field-cultivation trial of regenerated plants in two districts in Japan.
Field-cultivation of regenerated ipecac
23
30r---------~.-------------------------,
Material and Methods
25
Cultivation of regenerated plants Ipecac plants (Cephaelis ipecacuanha A. Richard) were clonally propagated by the method previously described (Ikeda et aI., 1988). The potted plants acclimatized in a phytotron (25°C, 16 h light/8 h dark, 80% relative humidity) for 1 month were transferred to the field in May (in 1987-1988) either soon (1M-plant) or after I-year pre-cultivation (1 Y-plant) in a greenhouse (20 to 35°C) and then cultivated under cheese cloth (50 % shading rate). Plants cultivated in the field of Tsukuba and Tanegashima were periodically collected to determine their growth and alkaloid contents.
20 ~ 15
10
~ E
10
f-
5 o+-------------------~~c-~~--~~ ·5~----------------------------------~ May Jun. Jul. Aug. Sep. Oct. Nov. Dec. Jan. Feb. Mar.
35
Extraction of alkaloids Plant materials (leaves and roots) were lyophilized and ground prior to extraction. The sample (ca. 10 mg) was accurately weighed and extracted according to the procedure described previously (Teshima et aI., 1984).
30 25 ~ ~
:J
20
10
iii 15 c.
E
HPLC analysis
f-
Alkaloid extracts were dissolved in an appropriate volume of MeOH and injected onto a TSK gel ODS-120A column (TOSOH, 4.6 i.d. X 250 mm), mobile phase acetonitrile-l0 mM sodium I-heptane sulfonate (pH 4.0, 33 : 67), flow rate 1.0 mLi min, UV detection at 285 nm at room temperature (Y oshimatsu and Shimomura, 1993).
10
o~----
May
Jun.
Jul.
Aug.
Sep.
Oct.
Nov.
Dec.
Jan.
Feb.
~
Mar.
Fig. 2: The atmospheric temperature in Tsukuba and Tanegashima. The data show monthly averages.
Results and Discussion
o
After a preliminary experiment in 1987, the cultivation experiment reported here was achieved in 1988. Tsukuba is located in the temperate zone (Lat. 36° N, Long. 140° E) and Tanegashima in the temperate zone close to the subtropics (Lat. 30 0 30' N, Long. 131 0 E) (Fig. 1). The atmospheric temperature in these districts is shown in Fig. 2.
______________________________
Jul.
2
3
4
5
7
8
EI
underground
9
10
F='
Aug.
Sep.
o
Oct. Nov.
FM (g)
aerial
t-----'
Dec.~
Long. i131 DE
I
. .
0
Long. ?40 E
~
i~!f jJ ~
~~
f)
v'.,
_______j____=_~~"~"b~L,t 36 'N i ~;~ (\t~--J(i, N.~·.J)G--y\ /. ~ )0' ~/ i
. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . +. . . . . . . . . . . Lat. 30 D30'N ~
"IS~\
! Tanegashima
i
Fig. 1: Map of Japan. The places where the regenerated plants were cultivated are indicated.
Jun.
Tanegashima
Jul. Aug.
Sep. Oct.
Nov. Dec. l'1ar. ,Jun.
Jul.
~--.----.----.----.----.----.----.-------.----.----.---_._--_._--_._--_._--_._--_._--_.
~
Greenhouse
Aug.~= ~ Sep. Oct.
Nov.~
r-----
Dec.~~~~~-----..J Fig. 3: Growth of 1Y-plants. The regenerated plants were pre-cultivated in a greenhouse for 1 year and then transplanted to a field in May (1988). The plants in the greenhouse were cultivated in pots (soil-sand-Ieaf mold,S: 1 : 1). Bars represent standard deviation of the mean, n = 2.
24
KAyO YOSHIMATSU, KAZUYA AOI, and KOICHIRO SHIMOMURA
Table 1: Growth characteristics of 1Y-plants cultivated in the field of Tanegashima station (n = 2 -10). Jui.
Aug.
Sep.
Oct.
Nov.
Dec.
plant height (em) standard deviation %C.V.
11.0 2.1 19.5
13.1 1.4 10.5
14.9 1.0 6.4
15.9 1.5 9.3
15.7 1.3 8.2
15.0 0.8 5.7
No. of leaves standard deviation %C.V.
7.6 2.1 27.8
9.2 2.3 25.5
9.3 1.0 11.2
8.3 0.8 9.8
7.8 0.5 6.5
7.5 0.7 9.4
max. leaf lengths (em) standard deviation %C.V.
6.4 1.1 17.6
7.8 0.9 11.7
8.5 1.0 11.9
8.6 1.0 11.4
8.9 1.7 19.0
7.8 0.6 7.3
max. leaf widths (em) standard deviation %C.V.
3.8 0.7 18.3
4.2 0.6 14.2
4.4 0.5 11.0
4.5 0.5 11.0
4.5 0.4 8.3
4.2 0.5 11.9
Remarkable differences of temperature between Tsukuba and Tanegashima occurred from October to March, and the minimum average monthly temperature in Tsukuba was below 0 °C from December to March. Growth of the 1Y-plants is shown in Fig. 3. The plants in the field of Tanegashima grew well from June to October compared with those in the field of Tsukuba and in the greenhouse. However, aerial parts were damaged significantly between January and February when minimum average monthly temperature was below 10°C. The plants in the field of Tsukuba did not grow well but the aerial parts remained alive until October. On the other hand, 1M-plants were damaged significantly after transplanting to the field, leading to the low ratio of establishment. Growth characteristics of 1Y-plants cultivated in the field of Tanegashima are presented in Table 1. Plant height, number of leaves, maximum leaf lengths, and maximum leaf widths increased from July to October and remained until December. The c.v.s of plant height, maximum leaf lengths and maximum leaf widths were relatively small during observation (below 20%), and the C.V.s of all parameters were smaller than 12 % from September to December. These results indicate that the regenerated plants grew homogeneously. The alkaloid contents of the 1Y-plants are shown in Fig. 4. The leaves contained lower amount of alkaloids than roots and rootlets. The alkaloid contents in the roots of 1Y-plants increased from June to December in Tanegashima, while they decreased in September and October in Tsukuba. The roots of 1Y-plants in a greenhouse demonstrated relatively stable alkaloid contents. The maximum alkaloid contents in the roots were obtained in December both in Tsukuba (2.4% DM cephaeline, 1.0% DM emetine) and Tanegashima (3.2 % DM cephaeline, 1.8 % DM emetine). The regenerated plants of C ipecacuanha clonally propagated through shoot-tip culture could be cultivated in the field when the atmospheric temperature was over 10°C. The variation of temperature significantly affected the growth and alkaloid contents of regenerated plants. Chatterjee et al. (1982) studied the field cultivation of this plant to provide information for economically feasible propagation
Alkaloid % DM
2
3
Jun·iii~~~TSU~kUbaS?l
Jul
0
cephaeline in rootlets
Aug.
EJ
cephaeline in roots
Sep.
Oct.
'
"
~
tj~~~c~=~h.< "'-' Dec. ~Hmffi§j~E~=====~=--1 Nov.
Jun.
_...._._._._-_._._._._.-
0
cephaeline in leaves
I!lI
emetine in rootlets
~
B
emetine in roots
Tanegashima emetine in leaves
-'-'-'-'-'---'-'-'--~'---'-'-'-'-'---'-'-'-'-'--
Fig. 4: Alkaloid contents in 1Y-plants. Cultivation conditions were the same as Fig. 3.
of ipecac in West Bengal and reported that ipecac seeds lost viability with storage; alkaloid content in the roots peaked in the third to forth year after germination. The regenerated plants in this study demonstrated homogeneous growth and a high alkaloid content within 2 years from transplantation to the soil. These results indicate that ipecac plants clonally propagated through shoot-tip culture are promising materials for cultivation trials in the field. Acknowledgement
Authors thank Mr. Kitazawa for technical assistance.
References CHATTERJEE, S. K., R. P. NANDI, and N. C. GHOSH: Cultivation and utilization of ipecac in West Bengal. In: ATAL, C. K. and B. M. KApUR (eds.): Cultivation and utilization of Medicinal plants, pp. 295-301, Regional Research Laboratory, Council of Scientific and Industrial Research, JAMMU-TAW!, India (1982). IKEDA, K., D. TESHlMA, T. AOYAMA, M. SATAKE, and K. SHIMOMURA: Clonal propagation of Cephaelis ipecacuanha. Plant Cell Rep. 7, 288-291 (1988). RAo, A. N. and S. K. LEE: Importance of tissue culture in tree propagation. In: FUJIWARA, A. (ed.): Proc. 5th IntI. Congo Plant Tissue and Cell Culture; Plant Tissue Culture 1982, pp. 715-718, The Japanese Association for Plant Tissue Culture, Japan (1982). TESHIMA, D., T. TSUCHIYA, T. AOYAMA, and M. HORIo!~A: Quantitative determination of main emetic components (eme-
Field-cultivation of regenerated ipecac tine and cephaeline) in ipecac. Iyakuhin kenkyu 15, 63-71 (1984). TREASE, G. E. and W. C. EVANS: Pharmacognosy 13th edition, pp. 595-599, Bailliere Tindal, London (1989).
25
K. and K. SHIMOMURA: Improved analysis of emetic alkaloids in tissue cultures and regenerates of Cephaelis ipeca· cuanha by ion pair high performance liquid chromatography. Phytochemical analysis 4, 217-219 (1993).
YOSHIMATSU,
Clonal Propagation of Cephaelis ipecacuanha (II): Characteristics of Regenerated Plants Field-Cultivated in two Districts KAyO
Y OSHIMATSU,
KAZUYA AOI*,
and KOICHIRO
SHIM OMURA
Tsukuba Medicinal Plant Research Station, National Institute of Health Sciences, 1 Hachimandai, Tsukuba, Ibaraki, 305 Japan
* Tanegashima Experiment Station of Medicinal Plants, National Institute of Health Sciences, 17019 Noma, NakatanecM, Kumage-gun, Kagoshima, 891-36 Japan
Received March 24, 1993 . Accepted December 10, 1993
Summary Ipecac plants, Cephaelis ipecacuanha A. Richard, were c10nally propagated through shoot-tip culture and field-cultivated at Tsukuba (Lat. 36° N, Long. 140° E) and Tanegashima (Lat. 30° 30' N, Long. 131 °E) in Japan. Growth and alkaloid content were periodically measured, and influence of environmental conditions was investigated. In Tanegashima, plants grew gradually from June to October, showing homogeneous growth of aerial parts. However the aerial parts were significantly damaged between January and February when minimum average monthly temperature was below 10°C. In Tsukuba, the plants did not grow well, but the aerial parts were alive until October. The alkaloid contents in the roots increased from June to December in Tanegashima, while in Tsukuba they decreased once in September and October. The maximum alkaloid content in the roots was observed in December, both in Tsukuba (2.4 % DM cephaeline, 1.0 % DM emetine) and Tanegashima (3.2 % DM cephaeline, 1.8 % DM emetine).
Key words: Cephaelis ipecacuanha, Rubiaceae, alkaloids, emetine, cephaeline, field-cultivation, regenerated plant, shoot-tip culture.
Abbreviations: HPLC = high performance liquid chromatography; FM = fresh mass; DM = dry mass; C.V. = coefficient of variation.
Introduction
Cephaelis ipecacuanha A. Richard (Family: Rubiaceae), native to the Amazon region and rich in alkaloids, is one of the important medicinal plants. Its dried roots and rhizomes, called ipecac, are commonly used as an expectorant in bronchitis, croup, asthma, and whooping cough, as an emetic in the cases of poisoning, and an amoebacide in amoebic dysentery (Trease and Evans, 1989). The supply of ipecac has mainly relied on collection of wild plants because only modest success of commercial cultivation has been achieved in Malaya and India (Trease and Evans, 1989). The best quality ipecac, Rio drug, is now be© 1994 by Gustav Fischer Verlag, Stuttgart
coming difficult to obtain because tropical forests are being gradually destroyed. Plant tissue culture has been considered as a promising tool for preservation of plant resources as well as the commercial mass propagation of plants (Rao and Lee, 1982). Recently we established micropropagation of ipecac plants through shoot-tip culture (Ikeda et al., 1988). However, for the tissue culture method to replace conventional methods, it is of importance to investigate the characteristics of growth and alkaloid content of the regenerated plants that are cultivated in the field. In this paper, we describe the field-cultivation trial of regenerated plants in two districts in Japan.
Field-cultivation of regenerated ipecac
23
30r---------~.-------------------------,
Material and Methods
25
Cultivation of regenerated plants Ipecac plants (Cephaelis ipecacuanha A. Richard) were clonally propagated by the method previously described (Ikeda et aI., 1988). The potted plants acclimatized in a phytotron (25°C, 16 h light/8 h dark, 80% relative humidity) for 1 month were transferred to the field in May (in 1987-1988) either soon (1M-plant) or after I-year pre-cultivation (1 Y-plant) in a greenhouse (20 to 35°C) and then cultivated under cheese cloth (50 % shading rate). Plants cultivated in the field of Tsukuba and Tanegashima were periodically collected to determine their growth and alkaloid contents.
20 ~ 15
10
~ E
10
f-
5 o+-------------------~~c-~~--~~ ·5~----------------------------------~ May Jun. Jul. Aug. Sep. Oct. Nov. Dec. Jan. Feb. Mar.
35
Extraction of alkaloids Plant materials (leaves and roots) were lyophilized and ground prior to extraction. The sample (ca. 10 mg) was accurately weighed and extracted according to the procedure described previously (Teshima et aI., 1984).
30 25 ~ ~
:J
20
10
iii 15 c.
E
HPLC analysis
f-
Alkaloid extracts were dissolved in an appropriate volume of MeOH and injected onto a TSK gel ODS-120A column (TOSOH, 4.6 i.d. X 250 mm), mobile phase acetonitrile-l0 mM sodium I-heptane sulfonate (pH 4.0, 33 : 67), flow rate 1.0 mLi min, UV detection at 285 nm at room temperature (Y oshimatsu and Shimomura, 1993).
10
o~----
May
Jun.
Jul.
Aug.
Sep.
Oct.
Nov.
Dec.
Jan.
Feb.
~
Mar.
Fig. 2: The atmospheric temperature in Tsukuba and Tanegashima. The data show monthly averages.
Results and Discussion
o
After a preliminary experiment in 1987, the cultivation experiment reported here was achieved in 1988. Tsukuba is located in the temperate zone (Lat. 36° N, Long. 140° E) and Tanegashima in the temperate zone close to the subtropics (Lat. 30 0 30' N, Long. 131 0 E) (Fig. 1). The atmospheric temperature in these districts is shown in Fig. 2.
______________________________
Jul.
2
3
4
5
7
8
EI
underground
9
10
F='
Aug.
Sep.
o
Oct. Nov.
FM (g)
aerial
t-----'
Dec.~
Long. i131 DE
I
. .
0
Long. ?40 E
~
i~!f jJ ~
~~
f)
v'.,
_______j____=_~~"~"b~L,t 36 'N i ~;~ (\t~--J(i, N.~·.J)G--y\ /. ~ )0' ~/ i
. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . +. . . . . . . . . . . Lat. 30 D30'N ~
"IS~\
! Tanegashima
i
Fig. 1: Map of Japan. The places where the regenerated plants were cultivated are indicated.
Jun.
Tanegashima
Jul. Aug.
Sep. Oct.
Nov. Dec. l'1ar. ,Jun.
Jul.
~--.----.----.----.----.----.----.-------.----.----.---_._--_._--_._--_._--_._--_._--_.
~
Greenhouse
Aug.~= ~ Sep. Oct.
Nov.~
r-----
Dec.~~~~~-----..J Fig. 3: Growth of 1Y-plants. The regenerated plants were pre-cultivated in a greenhouse for 1 year and then transplanted to a field in May (1988). The plants in the greenhouse were cultivated in pots (soil-sand-Ieaf mold,S: 1 : 1). Bars represent standard deviation of the mean, n = 2.
24
KAyO YOSHIMATSU, KAZUYA AOI, and KOICHIRO SHIMOMURA
Table 1: Growth characteristics of 1Y-plants cultivated in the field of Tanegashima station (n = 2 -10). Jui.
Aug.
Sep.
Oct.
Nov.
Dec.
plant height (em) standard deviation %C.V.
11.0 2.1 19.5
13.1 1.4 10.5
14.9 1.0 6.4
15.9 1.5 9.3
15.7 1.3 8.2
15.0 0.8 5.7
No. of leaves standard deviation %C.V.
7.6 2.1 27.8
9.2 2.3 25.5
9.3 1.0 11.2
8.3 0.8 9.8
7.8 0.5 6.5
7.5 0.7 9.4
max. leaf lengths (em) standard deviation %C.V.
6.4 1.1 17.6
7.8 0.9 11.7
8.5 1.0 11.9
8.6 1.0 11.4
8.9 1.7 19.0
7.8 0.6 7.3
max. leaf widths (em) standard deviation %C.V.
3.8 0.7 18.3
4.2 0.6 14.2
4.4 0.5 11.0
4.5 0.5 11.0
4.5 0.4 8.3
4.2 0.5 11.9
Remarkable differences of temperature between Tsukuba and Tanegashima occurred from October to March, and the minimum average monthly temperature in Tsukuba was below 0 °C from December to March. Growth of the 1Y-plants is shown in Fig. 3. The plants in the field of Tanegashima grew well from June to October compared with those in the field of Tsukuba and in the greenhouse. However, aerial parts were damaged significantly between January and February when minimum average monthly temperature was below 10°C. The plants in the field of Tsukuba did not grow well but the aerial parts remained alive until October. On the other hand, 1M-plants were damaged significantly after transplanting to the field, leading to the low ratio of establishment. Growth characteristics of 1Y-plants cultivated in the field of Tanegashima are presented in Table 1. Plant height, number of leaves, maximum leaf lengths, and maximum leaf widths increased from July to October and remained until December. The c.v.s of plant height, maximum leaf lengths and maximum leaf widths were relatively small during observation (below 20%), and the C.V.s of all parameters were smaller than 12 % from September to December. These results indicate that the regenerated plants grew homogeneously. The alkaloid contents of the 1Y-plants are shown in Fig. 4. The leaves contained lower amount of alkaloids than roots and rootlets. The alkaloid contents in the roots of 1Y-plants increased from June to December in Tanegashima, while they decreased in September and October in Tsukuba. The roots of 1Y-plants in a greenhouse demonstrated relatively stable alkaloid contents. The maximum alkaloid contents in the roots were obtained in December both in Tsukuba (2.4% DM cephaeline, 1.0% DM emetine) and Tanegashima (3.2 % DM cephaeline, 1.8 % DM emetine). The regenerated plants of C ipecacuanha clonally propagated through shoot-tip culture could be cultivated in the field when the atmospheric temperature was over 10°C. The variation of temperature significantly affected the growth and alkaloid contents of regenerated plants. Chatterjee et al. (1982) studied the field cultivation of this plant to provide information for economically feasible propagation
Alkaloid % DM
2
3
Jun·iii~~~TSU~kUbaS?l
Jul
0
cephaeline in rootlets
Aug.
EJ
cephaeline in roots
Sep.
Oct.
'
"
~
tj~~~c~=~h.< "'-' Dec. ~Hmffi§j~E~=====~=--1 Nov.
Jun.
_...._._._._-_._._._._.-
0
cephaeline in leaves
I!lI
emetine in rootlets
~
B
emetine in roots
Tanegashima emetine in leaves
-'-'-'-'-'---'-'-'--~'---'-'-'-'-'---'-'-'-'-'--
Fig. 4: Alkaloid contents in 1Y-plants. Cultivation conditions were the same as Fig. 3.
of ipecac in West Bengal and reported that ipecac seeds lost viability with storage; alkaloid content in the roots peaked in the third to forth year after germination. The regenerated plants in this study demonstrated homogeneous growth and a high alkaloid content within 2 years from transplantation to the soil. These results indicate that ipecac plants clonally propagated through shoot-tip culture are promising materials for cultivation trials in the field. Acknowledgement
Authors thank Mr. Kitazawa for technical assistance.
References CHATTERJEE, S. K., R. P. NANDI, and N. C. GHOSH: Cultivation and utilization of ipecac in West Bengal. In: ATAL, C. K. and B. M. KApUR (eds.): Cultivation and utilization of Medicinal plants, pp. 295-301, Regional Research Laboratory, Council of Scientific and Industrial Research, JAMMU-TAW!, India (1982). IKEDA, K., D. TESHlMA, T. AOYAMA, M. SATAKE, and K. SHIMOMURA: Clonal propagation of Cephaelis ipecacuanha. Plant Cell Rep. 7, 288-291 (1988). RAo, A. N. and S. K. LEE: Importance of tissue culture in tree propagation. In: FUJIWARA, A. (ed.): Proc. 5th IntI. Congo Plant Tissue and Cell Culture; Plant Tissue Culture 1982, pp. 715-718, The Japanese Association for Plant Tissue Culture, Japan (1982). TESHIMA, D., T. TSUCHIYA, T. AOYAMA, and M. HORIo!~A: Quantitative determination of main emetic components (eme-
Field-cultivation of regenerated ipecac tine and cephaeline) in ipecac. Iyakuhin kenkyu 15, 63-71 (1984). TREASE, G. E. and W. C. EVANS: Pharmacognosy 13th edition, pp. 595-599, Bailliere Tindal, London (1989).
25
K. and K. SHIMOMURA: Improved analysis of emetic alkaloids in tissue cultures and regenerates of Cephaelis ipeca· cuanha by ion pair high performance liquid chromatography. Phytochemical analysis 4, 217-219 (1993).
YOSHIMATSU,