Cytotoxic Alkaloids from the Roots of Tylophora atrofolliculata

• Abstract
• Introduction
• Materials and Methods
• General experimental procedures
• Plant material
• Extraction and Isolation
• Cytotoxicity Assay
• Results
• Discussion
• Acknowledgements
• References

Abstract

Four new phenanthroindolizidine alkaloids, tylophoridicines C-F, together with three known ones, R-(+)-deoxytylophorinidine, tylophorinine and tylophorinidine, were isolated from the roots of Tylophora atrofolliculata. The structures were determined on the basis of spectral evidence. These alkaloids exhibited cytotoxic activity in vitro on HCT-8 cell (with IC₅₀ values in the range 0.083 to 18.99 μM) and KB cell (in the range 3.56 to 18.22 μM) lines.

Introduction

The medicinal plant Tylophora atrofolliculata is widely distributed in Southwest of China. Its roots have long been used as a traditional medicine for the treatment of rheumatism [1]. The genus Tylophora is known to contain phenanthroindolizidine alkaloids that were proved to be cytotoxic due to the inhibition of protein and nucleic acid synthesis [2], [3], [4]. This encouraged us to perform more research on this type of compounds. In previous study, as a part work of our studies on antitumor bioactive constituents from traditional herbs, we discovered that the crude alkaloids extracted from the title plant displayed strong cytotoxic activity. Analysis of the crude alkaloids led to the isolation of four new phenanthroindolizidine alkaloids, tylophoridicines C-F (1 - 4), as well as three known ones R-(+)-deoxytylophorinidine (5), tylophorinine (6) and tylophorinidine (7). In this paper, we describe their isolation, elucidation and cytotoxic activity.

Materials and Methods

General experimental procedures

Melting points were measured on an XT₄ - 100X micromelting point apparatus and are uncorrected. Optical rotations were determined on a Perkin-Elmer 241 automatic digital polarimeter. The CD spectrum was obtained from a JOUAN Mark II spectropolarimeter. IR spectra were recorded using a Nicolet-Impact 400 IR spectrometer with KBr disks. UV spectra were obtained on a HP 8453 spectrophotometer. ¹H-, ¹³C-, DEPT, COSY, HMQC and HMBC NMR experiments were performed on an Inova 500 FT NMR spectrometer. TMS was used as internal standard. Mass spectra were recorded on an Autospec-UltimaETOF. Silica GF₂₅₄ for TLC and silica gel (160 - 200 mesh) for column chromatography were obtained from Qingdao Marine Chemical Company, Qingdao, People’s Republic of China. Solvents were analytical grade and purchased from Beijing Chemical Company, Beijing, People’s Republic of China.

Plant material

The roots of Tylophora atrofolliculata Mertcalf were collected from Nanning, Guangxi province, P. R. China in November 1999 and were identified by Prof. Shouyang Liu (Department of Pharmacognosy, Guangxi College of Chinese Traditional Medicine). A voucher specimen was deposited in the Herbarium of Institute of Materia Medica, Chinese Academy of Medical Sciences (No. C42).

Extraction and Isolation

The air-dried roots (7 kg) of Tylophora atrofolliculata were crushed and extracted three times with 95 % EtOH (20 L) under reflux. The combined ethanol extracts were evaporated under reduced pressure to yield a brownish viscous residue (791 g). A suspension of the resulting extract in hydrochloric acid (0.5 N, 1.5L) was filtered. The filtrate was made alkaline with NH₄OH (pH = 9.0) and extracted with CHCl₃ (4.3 L) until the water layer exhibited a negative Dragendorff test. The chloroform was removed in vacuum and an extract of 20.0 g crude total alkaloids was obtained, which was subjected to silica gel (1.0 kg) CC eluting with P.E.-CHCl₃ (20 : 1 5 L; 9 : 1 5 L; 4 : 1 5 L) and CHCl₃-MeOH (9 : 1 5 L; 4 : 1 5 L; 1 : 1 5 L; 0 : 1 3 L). Seven total fractions (A - G) were obtained from 110 fractions based on TLC behavior. The alkaloid-containing fractions (C-F) were identified by TLC using Dragendorff reagent. Fr. C (0.85 g) was re-subjected to silica gel (45 g) CC eluting with CH₂Cl₂ : CH₃OH (8 : 1, 1.5 L, 38 frs., 40 mL each) to give 7 sub-fractions (C₁-C₇) combined on TLC analysis. Compound 5 (121 mg) was obtained from Fr. C₃ and recrystallized from CHCl₃ : CH₃OH (8 : 1). Fr. D (1.95 g) was re-subjected to silica gel (100 g) CC eluting with CHCl₃:CH₃OH (12 : 1, 2.5 L, 58 frs., 40 mL each) to give 10 sub-fractions (D₁ - D₁₀) based on TLC. Compound 7 (1.12g) was crystallized from Fr. D₃ and recrystallized from CHCl₃ : CH₃OH (8 : 1). Fr. E (7.01 g) was further separated into 6 sub-fractions (E₁-E₆) after being re-subjected on silica gel (350 g) CC eluting with CHCl₃:MeOH (15 : 1, 4.5 L, 90 frs., 50 mL each) combined on TLC analysis. Compound 6 (3.52 g) was obtained from Fr. E₂-E₄ (4.27 g) and purified by crystallization from CHCl₃. Fr. F (2.53 g) was re-subjected to silica gel (100 g) CC eluting with CH₂Cl₂ : CH₃OH : NH₃OH (8 : 1 : 0.1, 2.5 L, 50 frs., 50 mL each) to obtained 12 sub-fractions (F₁-F₁₂) based on TLC analysis. Crystalline Fr. F₁₀ (610 mg) was recrystallized from CH₃OH to afford 1 (410 mg). Fr. F₉ (244 mg) was re-subjected to silica gel (15 g) CC eluting with CH₂Cl₂ : CH₃OH : NH₃OH (10 : 1 : 0.1, 0.5 L, 25 frs., 20 mL each) to obtained 8 sub-fractions (F₉a - F₉h) based on TLC analysis. Compound 3 (70 mg) was crystallized from Fr. F₉d. Fr. F₅ (93 mg) and Fr. F₃ (117 mg) were re-subjected to preparative TLC eluting with CH₂Cl₂ : CH₃OH : NH₃OH (10 : 1 : 0.1 or 12 : 1 : 0.1) to afford 2 (61 mg) and 4 (35 mg) respectively.

Tylophoridicine D (1): yellow amorphous powder; m. p. 295 - 310 °C; [α]_D ¹⁶: + 0.5° (c 1.0, MeOH); FABMS: m/z = 360 [M]⁺ (100), 344 [M - CH₄]⁺ (15); HRFAB-MS: m/z = 360.1585 (calcd. for C₂₃H₂₂NO₃ ⁺: 360.1599); UV (MeOH): λ_max (log ε) = 257 (4.68), 284 (4.56), 309 (sh, 4.06), 340 (3.46) nm; IR (KBr): ν_max = 3419, 1630, 1610, 1518, 1435, 1383, 1275 1211, 1167, 1045, 964, 839 cm^-1; NMR data: see Tables [1] and 2.

Tylophoridicine E (2): brown amorphous solid (from CHCl₃-MeOH); m. p. 225 - 226 °C; [α]_D ¹⁶: + 21.0° (c 0.74, CHCl₃); FABMS: m/z = 366 [M + 1]⁺ (40), 348 [M + 1-H₂O]⁺ (100), 296 [M - C₄H₇N]⁺ (17), 70 [C₄H₈N]⁺ (30); HRFABMS: m/z = 366.1713 [M+ 1]⁺ (calcd. for C₂₂H₂₃NO₄: 366.1705); UV (MeOH): λ_max (log ε) = 260 (4.63), 286 (4.35), 310 (sh, 3.88) nm; + NaOH: 256 (4.67), 295 (4.16), 331 (3.79) nm; IR (KBr): ν_max = 3427, 2954, 1618, 1514, 1469, 1263, 1203, 1165, 1032, 845 cm^-1; NMR data: see Tables [1] and 2; CD spectrum: see Fig. [3].

Tylophoridicine C (3): white powder (from CHCl₃); m. p. 234 - 237 °C; [α]_D ¹⁶: + 17.1° (c 0.37, CHCl₃); FABMS: m/z = 382 [M + 1]⁺ (60), 296 [M - C₄H₇NO]⁺ (100); HRFABMS: m/z = 382.1662 [M + 1]⁺ (calcd. for C₂₂H₂₃NO₅: 382.1654); UV (MeOH): λ_max (log ε) = 260 (4.63), 286 (4.35) nm; IR (KBr): ν_max = 3429, 2958, 1616, 1514, 1464, 1263, 1200, 1032, 970, 920 cm^-1; NMR data: see Tables [1] and 2.

Tylophoridicine F (4): white solid (from CHCl₃); m. p. 214 - 217 C; [α]_D ¹⁶: -11.0° (c 0.40, CHCl₃); HR-FABMS: m/z = 396.1798 (calcd. for C₂₂H₂₃NO₅: 396.1811); FABMS: m/z = 396, 309 (100); UV (MeOH): λ_max (log ε) = 260 (4.63), 286 (4.35) nm; IR (KBr): ν_max = 3440, 2960, 1621, 1514, 1233, 1210, 970, 922 cm^-1; NMR data see: Tables [1] and 2.

R-(+)-Deoxytylophorinidine (5): white needles (from CH₃COCH₃); [α]_D ¹⁸: + 176.0° (c 0.25, CHCl₃).

Tylophorinine (6): yellow powder (from CH₃COCH₃); [α]_D ²⁰: -12.3 ° (c 0.53, CHCl₃).

Tylophorinidine (7): white powder (from CHCl₃); [α]_D ²⁰: + 84.8 ° (c 0.84, CHCl₃).

Cytotoxicity Assay

We evaluated the cytotoxic activity of compounds 1 - 7 on KB and HCT-8 cell lines. Adriamycin was used as a positive control. The cells were continuously treated with the samples for 72 h. The supernatant was doffed off and 0.1 mL MTT (0.5 mg/mL in RPMI1640) was added after each well had been carefully washed with RPMI1640. The cell viabilities were measured with an MTT assay procedure (Table [3]) [5], [6] .

Results

Compounds 1 - 7 were identified as phenanthroindolizidine alkaloids on the basis of positive Dragendorff tests and similar NMR, UV spectral characters compared with those of reported compounds [7], [8].

Compound 1 was obtained as yellow amorphous powder. The molecular formula C₂₃H₂₂NO₃Cl was deduced from the HR-FAB-MS peak at m/z = 360.1585 (calcd. for C₂₃H₂₂NO₃ ⁺: 360.1599) and a positive silver chloride deposition reaction. The strong absorption bands in the UV spectrum at 340 (3.46) and 286 (4.56) nm were similar to those of dehydrotylophorine, which indicated that 1 was an aromatic alkaloid [9]. In the ¹H-NMR spectrum, three methoxy groups resonated at δ = 4.05 (3H, s), 4.04 (3H, s) and 4.03 (3H, s). The ¹H-NMR signals at 8.70 (1H, d, J = 9.2 Hz), 7.85 (1H, d, J = 2.5 Hz) and 7.34 (1H, dd, J = 9.2, 2.5 Hz) revealed a 1,2,4-trisubstituted phenyl ring (ring A). Ring B was deduced from the ¹H-NMR signals at 7.78 (1H, s), 7.99 (1H, s) and HMBC correlations of H-5 with C-6, C-7 and C-4a, H-8 with C-7 and C-8a. Ring D was established from ¹H-NMR signals at δ = 10.36 (1H, s), 8.98 (1H, s) and NOESY correlations of H-9 with H-8, H-14 with H-1 (see Fig. [1]). Three aliphatic carbon signals were assigned to ring E in the ¹³C-NMR spectrum. A full assignment of all ¹H- and ¹³C-NMR resonances of 1 was obtained from HMQC, HMBC and NOESY experiments. Therefore, the structure of compound 1 could be elucidated as 3,6,7-trimethoxy-9(10),13a(14)-dehydrophenanthroindolizidinium chloride. Govindachari inferred the presence of this compound during his isolation of the minor tertiary alkaloids of Tylophora asthmatica, but it could not be obtained as such [9].

Compound 2 was isolated as a brown amorphous solid. The HR-FAB-MS showed an [M]⁺ ion at m/z = 366.1713 (calcd. for C₂₂H₂₃NO₄: 366.1705) corresponding to the molecular formula C₂₂H₂₃NO₄. The UV absorptions at 286 (4.35), 310 (sh, 3.88) nm and the base peak at m/z = 296 [M - 69]⁺ in the mass spectrum indicated the existence of the phenanthroindolizidine skeleton. The bathochromic shifts at 256 (4.67), 295 (4.16) and 331 (3.79) nm in the UV spectrum when 1 N NaOH was added to the solution of 2 were assigned to an aromatic hydroxy group. In the ¹H-NMR spectrum, two methoxy group signals at δ = 3.98 (3H, s), δ = 3.88 (3H, s) and five aromatic proton signals from δ = 7.03 to 8.13 were observed. The signals at δ = 7.87 (1H, s), 7.03 (1H, s) were singlets assigned for the ring B. The other three aromatic proton signals at δ = 8.13 (1H, d, J = 9.0 Hz), δ = 7.10 (1H, dd, J = 9.0, 2.0 Hz) and δ = 7.90 (1H, d, J = 2.0 Hz) revealed the presence a 1,2,4-trisubstituted phenyl ring (ring A). The spectral data of ring D and ring E of 2 were similar to those of tylophorinidine [7], which suggested that position 14 was substituted by a hydroxy group. Therefore, compound 2 possessed the skeleton of a phenanthroindolizidine alkaloid with two methoxy moieties, a phenolic hydroxy group and an aliphatic hydroxy group. The positions of the methoxy moieties and the hydroxy groups were assigned with the aid of ¹H-¹HCOSY, HMQC, HMBC and NOESY experiments.

The CD spectrum of 2 displayed a negative Cotton effect in the 250 - 265 nm region (see Fig. [3]), which indicated the absolute configuration of C-13a was S [10]. In the ¹H-NMR spectrum, the H-14 signal appeared as a broad singlet, so that the dihedral angle between the 13a and 14 protons was close to 90°. Thus, the absolute configuration of C-14 was also S [11]. On the basis of these findings, the structure of 2 was identified as (13aS,14S)-3,14-dihydroxy-6,7-dimethoxyphenanthroindolizidine.

Compounds 3 and 7 were obtained as white solids. By comparing m. p., [α]_D, MS, UV and NMR data with those of references [11], [12], compound 7 was identified as tylophorinidine whose molecular formula was C₂₂H₂₃NO₄. The molecular formula of 3 was established as C₂₂H₂₃NO₅ by HR-FAB-MS at m/z = 382.1662 (calcd. for C₂₂H₂₃NO₅: 382.1654) suggesting 3 to be an N-oxide analogue of 7. Compound 3 showed similarities to 7 in its spectral data ([α]_D, UV, IR, MS and NMR). Comparing the proton signals of the two compounds, H-9, H-11, and H-13a of 3 showed downfield shifts (+ 0.68, + 1.39; + 0.28, + 1.48; + 1.08 ppm, respectively). In the ¹³C-NMR spectrum, downfield shifts were observed for the C-9, C-11, C-13a signals (+ 11.5, + 13.2, + 13.5 ppm, respectively) of 3. Therefore, compound 3 was characterized to be the N-oxide of 7 and established as (13aS,14S)-6,14-dihydroxy-3,7-dimethoxyphenanthroindolizidine N-oxide.

Compounds 4 and 6 were isolated as white and yellow solids, respectively. Compound 6 was identified as tylophorinine by comparing m. p. [α], MS, UV and NMR data with those of references [11], [12], [13]. The molecular formula of 6 was C₂₃H₂₅NO₄. The molecular formula C₂₃H₂₅NO₅ of 4 was deduced from HR-FAB-MS at m/z = 396.1798 (calcd. for C₂₃H₂₅NO₅: 396.1811) indicating compound 4 to be an N-oxide analogue of 6. There were very similar spectral data including [α]_D, UV, IR, MS and NMR between the two compounds except for downfield shifts (+ 1.48, 1.37; 1.06, 0.08; 1.35 ppm in the ¹H-NMR spectrum and + 12.2, + 14.9, + 14.7 ppm in the ¹³C-NMR spectrum, respectively) for positions 9, 11 and 13a of 4. Thus, compound 4 was elucidated to be the N-oxide of 6 and determined as (13aS,14R)-14-hydroxy-3,6,7-trimethoxyphenanthroindolizidine N-oxide.

Compound 5 was isolated as white needles and identified as R-(+)-deoxytylophorinidine by comparing m. p., [α], MS, UV and NMR data with those of reference [14].

Discussion

The chloride phenanthroindolizidine alkaloids had seldom been obtained from natural sources. As hydrochloric acid was used during the extraction, 1 was probably an artificial compound [9]. Although 3 and 4 were N-oxides of 7 and 6, respectively, they were not possible artifacts, because we had tried to get 3 and 4 through 7 and 6 but failed even when the latter two compounds were illuminated or heated in chloroform.

Phenanthroindolizidine alkaloids have revealed interesting biological properties including cytotoxic activity due to the inhibition of protein and nucleic acid synthesis [2], [3], [4]. Adriamycin was selected for the positive control upon initially consideration of the similar mechanism. According to a previous study [15], two aromatic rings (rendered slightly electron richer) and a nucleophilic element such as nitrogen at a certain distance from the aromatic rings were necessary for the cytotoxic activity of phenanthroindolizidine alkaloids. Compounds 1 - 7 were in accord with these qualifications so that all of the compounds showed cytotoxic activity. The activity of 2 was stronger than that of adriamycin on both KB and HCT-8 cells. Compound 5 displayed a stronger activity than adriamycin on KB cells. The IC₅₀ values of 7 for KB and HCT-8 cells were 5.48 and 3.56 μM. But when the unshared pair of electrons was oxidized to form an N-oxide (compound 3), the values changed to 25.00 and 11.54 μM, respectively. Accordingly, when the nitrogen atom of 6 was oxidized (compound 4), the IC₅₀ values on the two cell lines were changed from 6.60 μM on KB cells and 5.54 μM on HCT-8 cells to 18.99 μM on KB cells and 25.00 μM on HCT-8 cells. The change suggested that the unshared pair of electrons on nitrogen atom was at least in part responsible for the cytotoxic activity. This structure-activity relationship can also explain the relatively weak activity on KB cell of compound 1. Further tests on analogues are needed to confirm this conclusion.

Acknowledgements

The authors are grateful to the Department of Instrumental Analysis, Institute of Materia Medica, Chinese Academy of Medical Sciences and Peking Union Medical College for the measurements of IR, UV, NMR and mass spectra. We also thank Xiaoguang Chen's research group Institute of Materia Medica, Peking Union Medical College for the cell cultivation and bioactivity test.

References

• 1 Jiangsu New Medical College. Dictionary of Chinese Traditional Medicine. Shanghai; Shanghai Science and Technology Publishing House 1977: p 1747
  Search in Google Scholar
• 2 Hirokawa M, Yanauchi T, Honda K. Further investigation of phenanthroindolizidine alkaloids from Tylophora tanakae .  Chemical and Pharmaceutical Bulletin. 1998;  46 767-9
  PubMedSearch in Google Scholar
• 3 Narasimaha R K, Bhauacharya R K, Venkatachalam S R. Inhibition of thymidylate synthase and all growth by the phenanthroindolizidine alkaloids.  Cancer Letters. 1998;  128 183-8
  CrossrefPubMedSearch in Google Scholar
• 4 Gellert F, Rudzats R. Antileukemia activity of tylocrebrine.  Journal of Medical Chemistry. 1964;  7 361-2
  PubMedSearch in Google Scholar
• 5 Chen J J, Duh C Y, Chen I S. New tetrahydroprotoberberine N-oxide alkaloids and cytotoxic constituents of Corydalis tashiroi .  Planta Medica. 1999;  65 643-7
  Thieme ConnectPubMedSearch in Google Scholar
• 6 Mosmann T. Rapid colorimetric assay for cellular growth and survival: Application to proliferation and cytotoxicity assays.  Journal of Immunological Methods. 1983;  65 55-63
  CrossrefPubMedSearch in Google Scholar
• 7 Fumiko A, Yukiko I, Tatsuo Y, Keiichi H. Phenanthroindolizidine alkaloids from Tylophora tanakae .  Phytochemistry. 1995;  39 695-9
  CrossrefPubMedSearch in Google Scholar
• 8 Ali M, Butani K K. Alkaloids from Tylophora indica .  Phytochemistry. 1989;  28 3513-7
  CrossrefPubMedSearch in Google Scholar
• 9 Govindachari T R, Viswanathan N, Radhakrishnan J. Quaternary alkaloids from Tylophora asthmatica . Indian Journal of Chemistry 1973: 1255-1256
  Search in Google Scholar
• 10 Emery G, Richard R, John C C, Sushilk R, Ronald W W. Absolute configuration of tyliphoraine.  Australian Journal of Chemistry. 1978;  31 2095-7
  PubMedSearch in Google Scholar
• 11 Phillipson J D, Tezcan I, Hylands P J. Alkaloids of Tylophora species from Sri Lanka.  Planta Medica. 1974;  25 301-9
  PubMedSearch in Google Scholar
• 12 Mulchandani N B, Venkatakchalam S R. Alkaloids of Pergalaria pallida .  Phytochemistry. 1976;  15 561-3
  CrossrefPubMedSearch in Google Scholar
• 13 Govindachari T R, Viswanathan N, Radhakrishnan J. Minor alkaloids of Tylophora asthmatica revised structure of tylophorinidine.  Tetrahedron. 1973;  29 891-7
  CrossrefPubMedSearch in Google Scholar
• 14 Ying Z Y, Huang X S, Yu S S. Antitumor alkaloids Isolated from Tylophora ovata .  Acta Botanica Sinica. 2002;  44 349-3
  PubMedSearch in Google Scholar
• 15 Gupta R S, Krepinsky J J, Siminovitch L. Structural determinants responsible for the biological activity of (-)-emetine, (-)-cryptopleurine, and (-)-tylocrebrine: Structure-activity relationship among related compounds.  Molecular Pharmacology. 1980;  18 136-43
  PubMedSearch in Google Scholar

Prof. Dr. Shishan Yu

Institute of Materia Medica

Chinese Academy of Medical Sciences & Peking Union Medical College

No.1 Xiannongtan Street

Beijing 100050

People's Republic of China

Phone: +86-10-63165324

Fax: +86-10-63017757

Email: yushishan@imm.ac.cn

References

• 1 Jiangsu New Medical College. Dictionary of Chinese Traditional Medicine. Shanghai; Shanghai Science and Technology Publishing House 1977: p 1747
  Search in Google Scholar
• 2 Hirokawa M, Yanauchi T, Honda K. Further investigation of phenanthroindolizidine alkaloids from Tylophora tanakae .  Chemical and Pharmaceutical Bulletin. 1998;  46 767-9
  PubMedSearch in Google Scholar
• 3 Narasimaha R K, Bhauacharya R K, Venkatachalam S R. Inhibition of thymidylate synthase and all growth by the phenanthroindolizidine alkaloids.  Cancer Letters. 1998;  128 183-8
  CrossrefPubMedSearch in Google Scholar
• 4 Gellert F, Rudzats R. Antileukemia activity of tylocrebrine.  Journal of Medical Chemistry. 1964;  7 361-2
  PubMedSearch in Google Scholar
• 5 Chen J J, Duh C Y, Chen I S. New tetrahydroprotoberberine N-oxide alkaloids and cytotoxic constituents of Corydalis tashiroi .  Planta Medica. 1999;  65 643-7
  Thieme ConnectPubMedSearch in Google Scholar
• 6 Mosmann T. Rapid colorimetric assay for cellular growth and survival: Application to proliferation and cytotoxicity assays.  Journal of Immunological Methods. 1983;  65 55-63
  CrossrefPubMedSearch in Google Scholar
• 7 Fumiko A, Yukiko I, Tatsuo Y, Keiichi H. Phenanthroindolizidine alkaloids from Tylophora tanakae .  Phytochemistry. 1995;  39 695-9
  CrossrefPubMedSearch in Google Scholar
• 8 Ali M, Butani K K. Alkaloids from Tylophora indica .  Phytochemistry. 1989;  28 3513-7
  CrossrefPubMedSearch in Google Scholar
• 9 Govindachari T R, Viswanathan N, Radhakrishnan J. Quaternary alkaloids from Tylophora asthmatica . Indian Journal of Chemistry 1973: 1255-1256
  Search in Google Scholar
• 10 Emery G, Richard R, John C C, Sushilk R, Ronald W W. Absolute configuration of tyliphoraine.  Australian Journal of Chemistry. 1978;  31 2095-7
  PubMedSearch in Google Scholar
• 11 Phillipson J D, Tezcan I, Hylands P J. Alkaloids of Tylophora species from Sri Lanka.  Planta Medica. 1974;  25 301-9
  PubMedSearch in Google Scholar
• 12 Mulchandani N B, Venkatakchalam S R. Alkaloids of Pergalaria pallida .  Phytochemistry. 1976;  15 561-3
  CrossrefPubMedSearch in Google Scholar
• 13 Govindachari T R, Viswanathan N, Radhakrishnan J. Minor alkaloids of Tylophora asthmatica revised structure of tylophorinidine.  Tetrahedron. 1973;  29 891-7
  CrossrefPubMedSearch in Google Scholar
• 14 Ying Z Y, Huang X S, Yu S S. Antitumor alkaloids Isolated from Tylophora ovata .  Acta Botanica Sinica. 2002;  44 349-3
  PubMedSearch in Google Scholar
• 15 Gupta R S, Krepinsky J J, Siminovitch L. Structural determinants responsible for the biological activity of (-)-emetine, (-)-cryptopleurine, and (-)-tylocrebrine: Structure-activity relationship among related compounds.  Molecular Pharmacology. 1980;  18 136-43
  PubMedSearch in Google Scholar

Prof. Dr. Shishan Yu

Institute of Materia Medica

Chinese Academy of Medical Sciences & Peking Union Medical College

No.1 Xiannongtan Street

Beijing 100050

People's Republic of China

Phone: +86-10-63165324

Fax: +86-10-63017757

Email: yushishan@imm.ac.cn