Maytenus distichophylla and Salacia crassifolia: source of products with potential acetylcholinesterase inhibition

ABSTRACT

The phytochemical study of the extract leaves from Maytenus distichophylla Mart. and Salacia crassifolia (Mart. ex Schult.) G. Don, Celastraceae, resulted in the isolation of 3-oxofriedelane, 3β-hydroxyfriedelane, 3β,24-dihydroxyfriedelane, 3-oxo-28,29-dihydroxyfriedelane, two mixtures of pentacyclic triterpenes (α-amyrin with β-amyrin and 3β-stearyloxy-urs-12-ene with 3β-stearyloxy-olean-12-ene), 3β-palmityloxy-urs-12-ene, the steroid β-sitosterol and its glycosylated derivative β-glucosyl-β-sitosterol, tritriacontanoic acid and the natural polymer gutta percha. The chemical structures of these constituents were established by IR, ¹H and ¹³C NMR spectral data. Crude extracts, the mixtures of triterpenes and the isolated constituents were subjected to in vitro acetylcholinesterase inhibitory evaluation. Acetylcholinesterase inhibitory effect was observed for crude chloroform extract leaves from M. distichophylla (100%) and S. crassifolia (97.93 ± 5.63%) and for the triterpenes 3β,24-dihydroxyfriedelane (99.05 ± 1.12%), 3-oxo-28,29-dihydroxyfriedelane (90.59 ± 3.76%) and 3β-palmityloxy-urs-12-ene (97.93 ± 1.47%). The percent inhibitions induced by these natural products were very similar to those produced by physostigmine (93.94 ± 2.10%) a standard acetylcholinesterase inhibitor. Therefore, these results open perspectives for the use of these species as source of compounds with similar physostigmine pharmacological effect.

Keywords:
Celastraceae; Acetylcholinesterase; Natural products; Pentacyclic triterpenes; Physostigmine

Introduction

Neurodegenerative diseases result from chronic breakdown and progressive functional or structural loss of neurons, particularly those of the central nervous system (CNS). The accumulation of aggregated proteins at neurons has been correlated to these types of diseases (Park, 2010). The neurodegeneration process observed in Alzheimer′s disease (AD) has been characterized by progressive dementia and memory loss. Elevated levels of the peptide β-amyloid (Aβ) are associated with alterations of the synaptic function and neural network activity that probably underlies the cognitive deficits that occur in AD. Furthermore, the accumulation of this toxic peptide leads to deposition of Aβ into plaques and is thought to drive a pathologic cascade, which culminates in neuronal death (Cramer et al., 2012). The loss of cholinergic cells is accompanied by a decrease in the concentration of the acetylcholine (ACh). This endogenous compound is hydrolyzed by acetylcholinesterase (AChE), a hydrolytic enzyme of the serine class that is of major importance to physiology of the cholinergic synapses of the somatic system, the autonomic nervous system and the central nervous system (CNS) (Triggle et al., 1998). Therefore, one of the current accepted strategies in pharmacotherapy of AD has been the use of AChE inhibitors (Yang et al., 2012). As example, physostigmine (eserine) exerts a stereoselective inhibition of cholinesterase enzymes, such as AChE and butyrylcholinesterase (BuChE) by acting as a competitor or pseudosubstrate and transferring a carbamate residue to the enzyme's active site. Spontaneous hydrolysis regenerates the native enzyme and its function (Triggle et al., 1998).

The current drugs that act inhibiting AChE produce limited therapeutic results against AD, however, primarily provide a short-term alleviation of the symptoms, without blocking the progression of disease (Park et al., 2012). Until this moment, the development of more efficient AChE inhibitors, which act mainly in brain, has been considered as an effective approach to be applied for treating AD (Liu et al., 2013).

The nature is a rich source of biological and chemical diversity. Complex chemical structures isolated from natural products cannot be easily obtained by synthesis or semi synthesis in laboratories (Filho et al., 2006). The natural compounds, represented by the class of pentacyclic triterpenes (PCTT), are secondary plant metabolites that have a potential inhibitory property of AChE (Gurovic et al., 2010). The PCTT with skeleton lupane and friedelane can be included amongst the compounds to be used to treat CNS disorders observed in AD (Rodrigues et al., 2014).

The Celastraceae family represents a good source of PCTT that are of great interest, due to their wide range of biological activities (Silva et al., 2013). Species of this family, like Maytenus ilicifolia, have been used in traditional medicine of different regions of Brazil, for the treatment of gastric ulcers (De Andrade et al., 2007), inflammations, and diarrhea (Santos et al., 2007). In addition, the pharmacological potential of some Celastraceae species have being evidenced through its traditional use in Northeast of Brazil, as CNS stimulant, and to treat insomnia and migraine (Omena, 2009).

In the present work extracts and constituents from two species of the Celastraceae family, Maytenus distichophylla and Salacia crassifolia, were investigates in relation to the in vitro AChE inhibitory activity.

Materials and methods

¹H (400 MHz) and ¹³C (100 MHz) NMR experiments were carried out on a Bruker Avance DRX-400, operating at 300 K. The chemical shifts assignments (δ) were expressed in parts-per-million (ppm) and coupling constants (J) registered in Hertz (Hz). Tetramethylsilane (TMS) was used as internal standard (δ_H = δ_C = 0). The infrared spectra (IR) (1% KBr soln, 400–4000 cm^-1) were obtained on Shimadzu IR408 spectrometer. Melting points were determined on MQAPF-302 apparatus (Microquímica Equipamentos Ltda).

Column chromatography (CC) processes were performed using silica gel 60 [0.063–0.200 mm (70–230 mesh ASTM)], as stationary phase, and organic solvent pure, or in mixtures of crescent polarity, as mobile phase. Silica gel 60 (Merck) was used to prepare plates (0.25 mm) for analytic thin layer chromatography (TLC).

The leaves of Maytenus distichophylla Mart., Celastraceae, were collected in Jequié, Bahia, Brazil, and the species was identified by Dra. Guadalupe Licona de Macedo of Departamento de Botânica of Universidade Estadual do Sudoeste da Bahia (UESB), Brazil. A voucher specimen (No. HUESB 2093) was deposited in the Herbarium of Departamento de Botânica of UESB. The leaves of Salacia crassifolia (Mart. ex Schult.) G. Don, Celastraceae, were collected in Montes Claros, Minas Gerais, Brazil, and the species was identified by Dra. Maria Olívia Mercadante-Simões of Universidade Estadual de Montes Claros, Brazil. A voucher specimen (No. HBCB 144624) is preserved in the Herbarium of Instituto de Ciências Biológicas, UFMG. The plant materials were collected in accordance with authorization (Process: 010119/2014-0) to access to the genetic patrimony emitted by Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq), Brazil.

The leaves of M. distichophylla and S. crassifolia were dried at room temperature and fragmented on a mill, separately. Each powdered material was sequentially extracted with hexane, chloroform and ethyl acetate.

The chloroform extract (35.6 g) from M. distichophylla was subjected to silica gel CC, initially eluted with MeOH and then with CHCl₃, the last solvent yielded compound 13 (6 g). After removal the solvent, the fractions eluted with MeOH were submitted to successive silica gel CC, furnishing the constituents 7 and 8 as mixture (178 mg; hexane–CHCl₃ 70:30), 1 (55 mg; hexane–CHCl₃ 70:30), 2 (23 mg; hexane–CHCl₃ 60:40), 5 and 6 as mixture (35 mg; hexane–CHCl₃ 15:85), 10 (18 mg; hexane–EtOAc 95:05), 3 (272 mg; hexane–EtOAc 70:30), 12 (35 mg; CHCl₃–EtOAc 95:05), and 11 (87.3 mg; hexane–EtOAc 30:70).

The chloroform extract (44 g) from S. crassifolia was subjected to silica gel CC eluted with MeOH followed by CHCl₃ allowing the isolation of compound 13 (30 g). The fractions obtained with methanol, after the removal the solvent, were submitted to successive silica gel CC, furnishing the constituents, 9 (11.2 mg; hexane–CHCl₃ 80:20), 1 (10.2 mg; hexane–CHCl₃ 25:75), 2 (9.7 mg; hexane–CHCl₃ 15:85), 5 and 6 as mixture (3.7 mg; hexane–CHCl₃ 10:90), 10 (11.8 mg; hexane–CHCl₃ 5:95), 4 (24.4 mg; CHCl₃–EtOAc 27:75) and 12 (167.3 mg; CHCl₃–EtOAc 10:90).

The in vitro AChE inhibitory activity of extracts and constituents was evaluated using a 96-well microtiter plate following the Ellman′s method (Ellman et al., 1961). The buffer A (50 mM Tris–HCl, pH 8, containing 0.1 M NaCl and 0.02 M MgCl₂·6H₂O), B (50 mM Tris–HCl, pH 8, containing 0.1% bovine serum albumin), and C (50 mM Tris–HCl, pH 8) were prepared and used in this assay. The volumes of 25 µl of ACh iodide (15 mM in water), 125 µl of 5,5-dithiobis-(2-nitrobenzoic acid) (3 mM in buffer A), 50 µl of buffer B, and 25 µl of sample (10 mg/ml in MeOH diluted by 10 times with buffer C, providing a final concentration of 1 mg/ml) were added into each well of a 96-well microtiter plate. Instead of the addition of sample solution, 25 µl of buffer C was used to prepare the blank sample. The positive control physostigmine was prepared using similar procedure. Each assay was carried out in quintuplicate. The absorbance was measured at 405 nm every 60 s by eight times using an Elisa Thermoplate microplate reader. After addition of 25 µl of AChE solution (0.226 U/ml in buffer B), the absorbance was again measured every 60 s, for 10 times. The increase in absorbance relative to spontaneous hydrolysis of substrate was corrected by reaction rate variation before and after addition of the enzyme. The inhibition percentage was calculated by comparing the results produced by the samples and physostigmine, in relation to blank.

Results and discussion

Using phytochemical methods the following known compounds were isolated and identified of the leaves from M. distichophylla and S. crassifolia: 3-oxofriedelane (1) (Mahato and Kundu, 1994), 3β-hydroxyfriedelane (2) (Mahato and Kundu, 1994), 3β,24-dihydroxyfriedelane (3) (Costa and Carvalho, 2003) and 3-oxo-28,29-dihydroxyfriedelane (4) (Weeratunga et al., 1982), mixture of α-amyrin (5) and β-amyrin (8) (Mahato and Kundu, 1994), mixture of derivates of PCTT 3β-stearyloxy-urs-12-ene (6) (Miranda et al., 2006) and 3β-stearyloxy-olean-12-ene (9) (Vieira-Filho et al., 2003), 3β-palmityloxy-urs-12-ene (7) (Vieira-Filho et al., 2003), the steroid β-sitosterol (10) (Lendl et al., 2005) and its glycosylated derivative β-glucosyl-β-sitosterol (11) (Lendl et al., 2005), tritriacontanoic acid (12) (Hamdan et al., 2014), and the natural polymer gutta percha (13) (Oliveira et al., 2006). Even though occurring in distinct biomes, located about 1100 km far from one another, both species, M. distichophylla and S. crassifolia presents the compounds 1, 2, 5, 10, 12 and 13. This fact contributes to confirm that species of the Celastraceae family uses similar biosynthetic routes to produce its secondary metabolites. The main chemical shift assignments observed in the NMR spectra of compounds 1–13 are presented below.

In the ¹H NMR spectra of PCTT 1–4, a doublet signal at δ_H 0.87 (J = 6.80 Hz) was observed and it is in agreement to hydrogen of methyl C-23 of the friedelane skeleton. The ¹H and ¹³C NMR spectra of constituent 5 present signals at δ_H 5.13 (J = 3.60 Hz) and at δ_C 124.45 and δ_C 139.61, which are characteristic of ursane skeleton. The signal of a multiplet at 5.30, in the ¹H NMR, associated to the signals of carbinolic (80.60) and carbonyl (173.69) carbons, observed in the ¹³C NMR spectra of compounds 6 and 7, indicate an ester chain attached to C-3 of the PCTT. The presence of signals at δ_C 124.30 and δ_C 139.50 corroborates to identify these esters as being derivatives of 5. The signals at δ_C 121.60 and δ_C 145.20, correspondent to olefin carbon atoms, indicate compound 8 and 9 as oleanane derivatives. The signals at δ_C 80.61, correspondent to carbinolic carbon, and at δ_C 173.69, of carbonyl carbon, suggest compound 9 as an oleanane esther. In the ¹H NMR spectrum of compound 10, the signal at δ_H 5.35 was attributed to olefin hydrogen and a multiplet at δ_H 3.56 to hydrogen of hydroxylated carbon. The ¹³C NMR spectra of triterpenes 10 and 11 present similar profiles and the observed signal at δ_C 101.6 typical of glycoside, indicated 11 as a glycosylated steroid. The signals at δ_C 176.06 and at δ_C 14.12, observed in the ¹³C NMR spectrum of 12, were associated to carbonyl of carboxylic acid and the methyl group-end chain, respectively. Based on the signals of a triplet at δ_H 5.12 (J = 6.40 Hz), δ_C 134.90 and at δ_C 124.20, that are characteristic of olefin hydrogen, observed in the NMR spectra, together its appearance of a semi-solid compound 13 was identified as gutta percha. The constituents 1–13 isolated from leaves of M. distichophylla and S. crassifolia are in agreement with chemical profile of other species of the Celastraceae family (Silva et al., 2013). The compounds 3, 10, 11, and 12, and mixtures of 5 and 6, 7 and 8 from M. distichophylla, and all constituents from S. crassifolia have been isolated for the first time in these species.

Extracts and compounds from Celastraceae species have been showed potential in vitro AChE inhibitory activity (Alarcón et al., 2008, 2015; Yang et al., 2012; Rodrigues et al., 2014; Sousa et al., 2016). For example, sesquiterpene of β-agarofuran (epoxyeudesmane) skeleton, isolated from Maytenus disticha and Euonymus japonicas (Alarcón et al., 2008, 2015), pentacyclic triterpenes, from Maytenus sp. (Rodrigues et al., 2014) and flavonol triglycosides of leaves from Maytenus robusta, showed high AChE activity. Alarcón et al. (2008) showed that in polyhydroxy dihydroagarofuran sesquiterpenoid with a nucleus of the alatol-type are most active than others sesquiterpenes. On these compounds the presence of a free hydroxy group at C-15, and of the OAc (C-2) group next to OBz (C-1) could be responsible for the activity. These terpenoids possess mixed or uncompetitive mechanisms of inhibition of AChE, and were considered as models for the development of new naturally occurring drugs for management strategies for neurodegenerative diseases (Alarcón et al., 2015). Sousa et al. (2012) and Rodrigues et al. (2014) reported the phytochemical and the biological studies of Maytenus gonoclada Mart. and M. imbricata Mart. ex Reissek. In this works were observed high AChE inhibition induced by the pentacyclic triterpenes 3-oxo-11α-hydroxylup-20(29)-ene, 3-oxo-29-hydroxyfriedelane and 3,7-dioxofriedelane (Rodrigues et al., 2014) and for 3β-hydroxy-21β-H-hop-22(29)-ene, and 3β,11β-dihydroxyfriedelane (Sousa et al., 2012). Therefore, sesquiterpenes and the triterpenes showed AChE inhibitory activity, opening possibilities to the employment of these compounds as drug leads to be used in the treatment of Alzheimer's disease.

Herein, the chloroform extracts from leaves of M. distichophylla and S. crassifolia, and constituents 1–11 were assayed to in vitro AChE inhibition. For both chloroform extracts, and for 3β,24-dihydroxyfriedelane (3), 3-oxo-28,29-dihydroxyfriedelane (4) and 3β-palmityloxy-urs-12-ene (7) were observed AChE inhibitory activity (Table 1). The other constituents did not present AChE inhibition. The extract from M. distichophylla furnished the active compound 3 and the S. crassifolia extract the compounds 4 and 9. The extracts and compounds 3β,24-dihydroxyfriedelane (3) and 3β-palmityloxy-urs-12-ene (7) showed the same in vitro AChE inhibitory activity than physostigmine, used as positive control (Table 1). The results found in the present work confirmed that the PCTT have AChE inhibition property and open perspectives to the employment of these compounds in researches involving pharmacological activities similar to those presented by physostigmine.

Table 1
AChE inhibitory activity to chloroform extracts and compounds of the leaves from Maytenus distichophylla and Salacia crassifolia.

Samples	AChE activity
	Inhibition (%)	Standard deviation	Coefficient of variation
Chloroform extract from M. distichophylla	100	Nd	Nd
Chloroform extract from S. crassifolia	97.93	5.63	0.06
3β,24-Dihydroxyfriedelane (3)	99.05	1.12	0.01
3-Oxo-28,29-dihydroxyfriedelane (4)	90.59	3.76	0.04
3β-Palmityloxy-urs-12-ene (7)	94.90	1.47	0.02
Physostigmine (Eserine)a	93.94	2.10	0.02

• Nd, not detected.
• ^a
  Positive control.

• Ethical disclosures
  Protection of human and animal subjects. The authors declare that no experiments were performed on humans or animals for this study.
  Confidentiality of data. The authors declare that they have followed the protocols of their work center on the publication of patient data.
  Right to privacy and informed consent. The authors declare that no patient data appear in this article.

Acknowledgments

The authors thank to FAPEMIG, CAPES and CNPq for financial support.

References

• Alarcón, J., Astudillo, L., Gutierrez, M., 2008. Inhibition of acetylcholinesterase activity by dihydroagarofuran sesquiterpenes isolated from Chilean Celastraceae. Z. Naturforsch. 63c, 853-856.
• Alarcón, J., Céspedes, C.L., Muñoz, E., Balbontin, C., Valdes, F., Gutierrez, M., Astudillo, L., Seigler, D.S., 2015. Dihydroagarofuranoid sesquiterpenes as acetylcholinesterase inhibitors from Celastraceae plants: Maytenus disticha and Euonymus japonicus J. Agric. Food Chem. 63, 10250-10256.
• Costa, P.M., Carvalho, M.G., 2003. New triterpene isolated from Eschweilera longipes (Lecythidaceae). An. Acad. Bras. Cienc. 75, 21-25.
• Cramer, P.E., Cirrito, J.R., Wesson, D.W., Lee, C.Y.D., Karlo, J.C., Zinn, A.E., Casali, B.T., Restivo, J.L., Goebel, W.D., James, M.J., Brunden, K.R., Wilson, D.A., Landreth, G.E., 2012. ApoE-directed therapeutics rapidly clear β-amyloid and reverse deficits in ad mouse models. Science 335, 1503-1506.
• De Andrade, S.F., Lemos, M., Comunello, E., Noldin, V.F., Chechinel-Filho, V., Niero, R., 2007. Evaluation of the antiulcerogenic activity of Maytenus robusta (Celastraceae) in different experimental ulcer models. J. Ethnoparmacol. 113, 252-257.
• Ellman, G.L., Courtney, K.D., Andres, V., Featherstone, R.M., 1961. A new and rapid colorimetric determination of acetylcholinesterase activity. Biochem. Pharmacol. 7, 88-95.
• Filho, J.M.B., Medeiros, K.C.P., Diniz, M.F., Batista, L.M., Athayde-Filho, P.F., Silva, M.S., Cunha, E.V.L., Almeida, J.R.G.S., Quintans-Junior, L.J., 2006. Natural products inhibitors of the enzyme acetylcholinesterase. Rev. Bras. Farmacogn. 16, 258-285.
• Gurovic, M.S.V., Castro, M.J., Richmond, V., Faraoni, M.B., Maier, M.S., Murray, A.P., 2010. Triterpenoids with acetylcholinesterase inhibition from Chuquiraga erinacea D. Don. subsp. erinacea (Asteraceae). Planta Med. 76, 607-610.
• Hamdan, D., Wink, M., El-Shazly, A., 2014. Secondary metabolites isolated from dichloromethane fraction of rough lemon stem and hepatoprotective effect of limonianin. Br. J. Pharm. Res. 4, 1963-1975.
• Lendl, A., Werner, I., Glasl, S., Kletter, C., Mucaji, P., Presser, A., Reznicek, G., Jurenitsch, J., Taylor, D.W., 2005. Phenolic and terpenoid compounds from Chione venosa (sw.) Urban var. venosa (Bois Bandé). Phytochemistry 66, 2381-2387.
• Liu, J.-Q., Peng, X.-R., Li, X.-Y., Li, T.-Z., Zhang, W.-M., Shi, L., Han, J., Qiu, M.-H., 2013. Norfriedelins A–C with acetylcholinesterase inhibitory activity from acerola tree (Malpighia emarginata). Org. Lett. 15, 1580-1583.
• Mahato, S.B., Kundu, A.P., 1994. ¹³C NMR spectra of pentacyclic triterpenoids – a compilation and some salient features. Phytochemistry 37, 1517-1575.
• Miranda, R.R.S., Silva, G.D.F., Duarte, L.P., Fortes, I.C.P., Vieira-Filho, S.A., 2006. Structural determination of 3β-stearyloxy-urs-12-ene from Maytenus salicifolia by 1D and 2D NMR and quantitative ¹³C NMR spectroscopy. Magn. Reson. Chem. 44, 127-131.
• Oliveira, D.M., Silva, G.D.F., Duarte, L.P., Vieira-Filho, S.A., 2006. Chemical constituents isolated from roots of Maytenus acanthophylla Reissek (Celastraceae). Biochem. Syst. Ecol. 34, 661-665.
• Omena, M.R.L.A., 2009. Ensaio etnofarmacológico de espécies vegetais com ação no sistema nervoso central, originárias do bioma caatinga. Saúde Ambient. Rev. 2, 92-95.
• Park, S.-Y., 2010. Potencial therapeutic agents against Alzheimer's disease from natural sources. Arch. Pharm. Res. 33, 1589-1609.
• Park, S.J., Jung, J.M., Lee, H.E., Lee, Y.W., Kim, D.H., Kim, J.M., Hong, J.G., Lee, C.H., Jung, I.H., Cho, Y.-B., Jang, D.S., Ryu, J.H., 2012. The memory ameliorating effects of INM-176, an ethanolic extract of Angelica gigas, against scopolamine- or Aβ(1-42)-induced cognitive dysfunction in mice. J. Ethnopharmacol. 142, 611-620.
• Rodrigues, V.G., Silva, F.C., Duarte, L.P., Takahashi, J.A., Matildes, B.L.G., Silva, G.D.F., Silva, R.R., Vieira-Filho, S.A., 2014. Pentacyclic triterpenes from Maytenus genus as acetylcholinesterase inhibitors. Int. J. Pharm. Pharm. Sci. 6, 918-920.
• Santos, V.L., Costa, V.B.M., Agra, M.F., Silva, B.A., Batista, L.M., 2007. Pharmacological studies of ethanolic extracts of Maytenus rigida Mart (Celastraceae) in animal models. Rev. Bras. Farmacogn. 17, 336-342.
• Silva, F.C., Oliveira, M.L.G., Rodrigues, V.G., Carvalho, S.M., Duarte, L.P., Silva, G.D.F., Miranda, R.R.S., Figueiredo, R.C., Moraes, J.C., Vieira-Filho, S.A., 2013. Triterpenes from Maytenus gonoclada and their attractive effects on Tenebrio molitor Chem. Nat. Compd. 49, 571-574.
• Sousa, G.F., Duarte, L.P., Alcântara, A.F.C., Silva, G.D.F., Vieira-Filho, S.A., Silva, R.R., Oliveira, D.M., Takahashi, J.A., 2012. New triterpenes from Maytenus robusta: structural elucidation based on NMR experimental data and theoretical calculations. Molecules 17, 13439-13456.
• Sousa, G.F., Aguilar, M.G., Takahashi, J.A., Alves, T.M.A., Kohlhoff, M., Vieira Filho, S.A., Silva, G.D.F., Duarte, L.P., 2016. Flavonol triglycosides of leaves from Maytenus robusta with acetylcholinesterase inhibition. Phytochem. Lett. 19, 34-38.
• Triggle, D.J., Mitchell, J.M., Filler, R., 1998. The pharmacology of physostigmine. CNS Drug Rev. 4, 87-136.
• Vieira-Filho, S.A., Duarte, L.P., Silva, G.D.F., Howarth, O.W., Lula, I.S., 2003. 3β-(Stearyloxy) olean-12-ene from Austroplenckia populnea: structure elucidation by 2D-NMR and quantitative ¹³C-NMR spectroscopy. Helv. Chim. Acta 86, 3445-3449.
• Weeratunga, G., Kumar, V., Sultanbawa, M.U.S., Balasubramaniam, S., 1982. 28,29-Dihydroxyfriedelan-3-one, a friedelane with two oxygenated methyl groups, from Elaeodendron balae (Celastraceae). J. Chem. Soc. Perkin Trans. 1, 2457-2459.
• Yang, Z.-D., Duan, D.-Z., Xue, W.-W., Yao, X.-J., Li, S., 2012. Steroidal alkaloids from Holarrhena antidysenterica as acetylcholinesterase inhibitors and the investigation for structure–activity relationships. Life Sci. 90, 929-933.

Publication Dates

• Publication in this collection
  Jul-Aug 2017

History

• Received
  21 July 2016
• Accepted
  16 Dec 2016