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Chemical constituents of the

hemiparasitic plant Phoradendron
brachystachyum DC Nutt (Viscaceae)
a b
Sugey López-Martínez , Gabriel Navarrete-Vázquez , Samuel
b a a
Estrada-Soto , Ismael León-Rivera & María Yolanda Rios
a
Centro de Investigaciones Químicas, Universidad Autónoma del
Estado de Morelos, Avenida Universidad 1001, Col. Chamilpa,
Cuernavaca, Morelos 62209, México
b
Facultad de Farmacia, Universidad Autónoma del Estado de
Morelos, Avenida Universidad 1001, Col. Chamilpa, Cuernavaca,
Morelos 62209, México
Version of record first published: 23 Feb 2012.

To cite this article: Sugey López-Martínez , Gabriel Navarrete-Vázquez , Samuel Estrada-Soto ,

Ismael León-Rivera & María Yolanda Rios (2013): Chemical constituents of the hemiparasitic plant
Phoradendron brachystachyum DC Nutt (Viscaceae), Natural Product Research: Formerly Natural
Product Letters, 27:2, 130-136

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 Natural Product
Natural Research2013
ProductResearch,
Vol. 27,
2012, No.iFirst
1–7, 2, 130–136, http://dx.doi.org/10.1080/14786419.2012.662646

Chemical constituents of the hemiparasitic plant Phoradendron

brachystachyum DC Nutt (Viscaceae)
Sugey López-Martı́neza, Gabriel Navarrete-Vázquezb, Samuel Estrada-Sotob,
Ismael León-Riveraa and Marı́a Yolanda Riosa*
a
Centro de Investigaciones Quı´micas, Universidad Autónoma del Estado de Morelos,
Avenida Universidad 1001, Col. Chamilpa, Cuernavaca, Morelos 62209, Me´xico;
b
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Facultad de Farmacia, Universidad Autónoma del Estado de Morelos, Avenida Universidad 1001,
Col. Chamilpa, Cuernavaca, Morelos 62209, Me´xico
(Received 20 April 2011; final version received 10 November 2011)

Phoradendron brachystachyum is a hemiparasitic plant widely distributed in

México that belongs to the Viscaceae family. It has been commonly used in folk
medicine as a substitute for the European mistletoe. In this chemical study,
morolic acid was isolated as the major component (47.54% of the total
composition of acetone extract) of this plant. In addition, 19 known compounds
were identified: �-sitosteryl and stigmasteryl linoleates, �-sitosterol, stigmasterol,
triacontanol, squalene, �- and �-amyrin, lupeol, lupenone, betulin aldehyde,
betulon aldehyde, oleanolic aldehyde, betulinic acid, betulonic acid, moronic acid,
morolic acid, oleanolic acid, flavonoids acacetin and acacetin 7-methyl ether.
There have been no previous reports in the literature on the chemical composition
of this potential natural source of hypoglycaemic and antihypertensive
compounds.
Keywords: Phoradendron; Viscaceae; mistletoe; morolic acid; triterpenes; TLC

1. Introduction
For thousands of years, medicine and natural products have been closely linked through
the use of traditional medicines and natural poisons. Clinical, pharmacological and
chemical studies of these traditional medicines, derived predominantly from plants, were
the basis of most early medicines.
In México, there are 6.5 million young diabetic people. Today, one in three adolescents
is overweight or obese, is dependent on injected insulin, or has hypertension, afflictions
usually confined to adulthood. Sedentary lifestyle, excessive food intake and insufficient
consumption of natural nutrients are the main factors causing young people to acquire
these pathologies. According to the National Health Survey 2007, México has the second
highest obesity rate in the world. In México City, 7.2% of the population suffers from this
pathology, leading to degenerative, incurable diseases such as type 2 diabetes, arterial
hypertension and renal damage. In México, the Phoradendron genus has a very wide
geographic distribution. The plants belonging to this genus are known as ‘corpo’,
‘muérdago’, ‘mistletoe’, ‘injerto’ and ‘yerba pajarito’, among others. These plants
have been used in different parts of the world as a traditional medicine for diabetes

*Corresponding author. Email: myolanda@uaem.mx

ISSN 1478–6419 print/ISSN 1478–6427 online

*Corresponding
� author. Email: myolanda@uaem.mx
2012 Taylor & Francis
http://dx.doi.org/10.1080/14786419.2012.662646
© 2013 Taylor & Francis
http://www.tandfonline.com
 2 S. López-Martı´nez et al. Natural Product Research   131

(Andrade-Cetto & Heinrich, 2005), hypertension (Varela et al., 2004), gastric cytoprotec-
tion (Gonzales, Iglesias, Carretero, & Villar, 2000) and immunomodulation treatment
(Varela et al., 2004). Extracts of mistletoe have been shown to kill cancer cells in the
laboratory and to stimulate the immune system (Hajto & Lanzrein, 1986). Three
components of mistletoe, lectins, viscotoxins and alkaloids, may be responsible for its
biologic effects.
Phoradendron brachystachyum is a hemiparasitic plant constituting an ever green
plague (Chazaro et al., 1992). In México City, they grow on trees in forests, parks and
gardens (Rivera & Espinosa, 2007), putting the lives of host trees at risk; thus, the
conservation of the host trees requires extensive gardening work. Tlalpan, Milpa Alta,
Coyoacán and Xochimilco are zones of México City with a high presence of Phoradendron
species, with P. brachystachyum as the predominant species; annually, these plants produce
tons of foliage that is considered to be waste material.
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Previously, chemical analysis of P. reichenbachianum showed that squalene, glycerol

trilinoleate, �-germanicol, lupeol, �-sitosterol, betulin aldehyde, betulon aldehyde,
�-sitosteril glucopyranoside, moronic, morolic and 3,4-seco-olean-18-en-3,28-dioic acids
are the chemical constituents of the acetone extract of this plant. Moronic acid (21.02%)
and morolic acid (0.53%) are the major constituents (Rios, Salinas, & Villarreal, 2001) and
their presence and amount in P. reichenbachianum as been associated with the antidiabetic
activity attributed to this medicinal plant (Ramı́rez-Espinosa et al., 2011). Anti-
inflammatory, anti-HIV, anti-HSV (anti-herpes simplex viruses-1) and antimicrobial are
other important activities associated to these natural triterpenes. Even though the
Loranthaceae represents one of the most extensive families of higher plants (75 genera,
1300 species), its members have been poorly investigated from chemical as well as
pharmacological perspectives. Little is known on the chemical composition of
Phoradendron genus plants, and to our knowledge, this is the first phytochemical analysis
to P. brachystachyum.

2. Results and discussion

After primary percolation and successive chromatography, isolates from the acetone
extract of the aerial parts of P. brachystachyum (Figure S1) were identified as 20 chemical
constituents. These include: 12 triterpenes [�-amyrin (1), �-amyrin (2) (Heupel, 1985),
oleanolic aldehyde (3) (Hota & Bapuji, 1994), oleanolic acid (4) (Seebacher, Simic, Weis,
Saf, & Kunert, 2003), moronic acid (5) (Cao et al., 2004), morolic acid (6) (Zhang, Hao,
Liu, Zhang, & Sun, 2009), lupeol (7) (Burns, Reynolds, Buchanan, Reese, & Enriquez,
2000), lupenone (8) (Carpenter, Sotheeswaran, Sultanbawa, & Ternai, 1980), betulin
aldehyde (9) (Hata, Hori, & Takahashi, 2002), betulon aldehyde (10) (Bohlmann et al.,
1977), betulinic acid (11) (Sholichin, Yamasaki, Kasai, & Tanaka, 1980) and betulonic
acid (12) (González, Amaro, Fraga, & Luis, 1983)] (Figure 1), two linear hydrocarbons
[squalene and triacontanol], four sterols [�-sitosteryl linoleate, stigmasteryl linoleate,
stigmasterol and �-sitosterol] and two flavonoids [acacetin and acacetin 7-methyl ether].
Pentacyclic triterpenes with a lupane and oleanane skeleton are the main components
of this extract, isolated as both C3�-hydroxy- and C3-oxo-types. Compounds 2–4
correspond to the C3�-hydroxy-D12,13-oleanane skeleton possessing methyl, aldehyde and
carboxylic acid residues at C28, respectively (Figure 1). Compounds 5 and 6 correspond to
C3�-hydroxy- and C3-oxo-D18,19-oleanane. On the other hand, triterpenes 7–12 include
both C3�-hydroxy- and C3-oxo-lupane, possessing methyl, aldehyde and carboxylic acid
residues at C28.
 132   S. López-Martínez et al. Natural Product Research 3

R1

R2

R3 R1

R4 R2
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1 R1=H; R2=R3=CH3; R4= β-OH; ∆12,13 7 R1=CH3; R2=β-OH

2 R1=R3=CH3; R2=H; R4= β-OH; ∆12,13 8 R1=CH3; R2=O
3 R1=CH3; R2=H; R3=CHO; R4=β-OH; ∆12,13 9 R1=CHO; R2=β-OH
4 R1=CH3; R2=H; R3=COOH; R4=β-OH; ∆12,13 10 R1=CHO; R2=O
5 R1=CH3; R2=H; R3=COOH; R4=β-OH; ∆18,19 11 R1=COOH; R2=β-OH
12 R1=COOH; R2=O
6 R1=CH3; R2=H; R3=COOH; R4=O; ∆18,19

Figure 1. Oleanane and lupane triterpenes from P. brachystachyum.

29

30

11
25 26
1 28
2

27
HO

23 24

Figure 2. Selected cross peaks in HMBC of �-amyrin (1).

Compounds 1 and 2 could be differentiated by the following spectroscopic evidences

(Table S1, Figures S2–S10): (1) the downfield shifts at 13C NMR of C-13 (D�C 143.38–
139.79 ¼ 3.59) by the presence of a C-30 methyl group at C-19 for compound 1; (2) an
upfield shift for C-18, C-20 and C-22 (D�C 48.60–59.27 ¼ � 10.67, D�C 31.30–39.82 ¼ �8.52
and D�C 37.32–41.73 ¼ �4.41, respectively) and (3) an upfield shift for C-19 and C-21 (D�C
47.89–39.82 ¼ 8.07 and D�C 36.75–31.46 ¼ 5.29, respectively) at E ring region of compound
1. For �-amyrin (1), HMQC experiment showed cross peaks relating H–C as in Table S1.
Selected HMBC cross peaks for compound 1 are illustrated in Figure 2. Of this manner, H23
(�H 0.79) and H24 (�H 0.95) showed cross peaks with C3 (�C 79.28), C4 (�C 39.87) and C5 (�C
55.39); H25 (�H 1.00) with C5 and C9 (�C 47.92); H26 (�H 0.91) with C9; H27 (�H 1.07) showed
 4 S. López-Martı´nez et al. Natural Product Research   133

cross peaks with C8 (�C 40.22), C14 (�C 42.28) and C15 (�C 28.34); H28 (�H 1.01) with C17 (�C
33.97) and C22 (�C 41.73) and finally, H29 showed cross peak with C20 (�C 39.82) while H30
with C18 (�C 59.27) and C19 (�C 39.82) (Figures S6–S8 from supporting material).
Compounds 2–4 show characteristic signals for the vinyl carbons C-12 and C-13
approximately at �C 124 and 144, respectively (Seebacher et al., 2003), whereas compounds
5–6 show resonance for the vinyl carbons around of C-18 and C-19 at �C 137 and 133,
respectively (Table S1 and Figures S11–S14, Rios et al., 2001; Zhang et al., 2009). For the
last two compounds, C-16 shows an upfield shift near 10 ppm (for example D�C 23.80–
33.60 ¼ �9.8 for compound 6 with respect to compound 4). Finally, the 1H NMR of
compounds 2–4 show a doublet of doublet signal at 3.3 ppm for H-18 and a doublet of
doublet signal with �H around 5.5 ppm for H-12 (Seebacher et al., 2003). Compounds 5–6
show the vinyl hydrogen (H-19) resonant as a singlet near 5.2 ppm (Rios et al., 2001;
Zhang et al., 2009).
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Morolic acid (6) is the major component of the acetone extract obtained from all
percolation processes. This compound was obtained in an amount so important, that it
spontaneously precipitates at each eluted fraction, to a total of 28.53 g, which represents
47.54% of the total extract weight, regardless of the amount remaining in the mother
liquor. This fact is very important because it suggests that the presence of this compound
directly correlates with the antihypertensive and the hypoglycaemic activities reported for
this species, as has been previously demonstrated for this compound isolated from
P. reichenbachianum (Ramı́rez-Espinosa et al., 2011). Additionally, compound 6 possesses
probed anti-inflammatory activity that is effective at relieving mouse ear inflammation
induced by 12-O-tetradecanoylphorbol 13-acetate and at inhibiting phospholipase A2
(PLA2) induced foot paw oedema. PLA2 is strongly associated with the control and
regulation of inflammatory processes involving arachidonic acid metabolism and
phospholipid turnover (Paduch, Kandefer-Szerszen, Trytek, & Fiedurek, 2007).
Furthermore, it significantly reduced the swelling in a chronic in vivo model (Adams,
Berset, Kessler, & Hamburger, 2009). Interestingly, dimethylsuccinate derivative of
morolic acid showed antiretroviral activity in a novel single-cycle replication assay,
probably inhibiting human immunodeficiency virus replication (anti-HIV-1 activity) by
blocking CA-SP1 processing (Dorr, Yemets, Kolomitsyna, Krasutsky, & Mansky, 2011).
On other hand, moronic acid (5) exhibited potent in vitro anti-herpes simplex viruses-1
(HSV-1) activity (EC50 ¼ 3.9 mg mL�1) and has been orally administered to mice
cutaneously infected with these viruses to study its in vivo efficacy. It significantly
retarded the development of skin lesions of infected mice (Kurokawa et al., 1999).
Compound 5 also showed significant anti-HIV activity with an EC50 of 0.1 mg mL�1 in H9
lymphocytes (Ito et al., 2001). This natural oleanane triterpene has low toxicity (Qian
et al., 2010) and seems to have antimicrobial activity against Escherichia coli,
Staphylococcus species and Ozava muconata (Hostettmann-Kaldas & Nakanishi, 1979).
Compounds 5 and 6 have been isolated from other natural sources (Lee, 2004).
However, only the species in the Phoradendron genus biosynthesised significant amounts of
these natural triterpenes, for example, P. brachystachyum, a plant specialised in the
biosynthesis of morolic acid (6) and P. reichenbachianum, which has moronic acid (5) as its
major component (Rios et al., 2001).

3. Experimental
3.1. General procedures
Compounds were isolated by primary percolation and successive open column chroma-
tography (CC). The isolation procedures and purity of compounds were monitored by thin
 134   S. López-Martínez et al. Natural Product Research 5

layer chromatography (TLC), observing the plates under UV light and then spraying the
plates with (NH4)4Ce(SO4)4 in 2 N H2SO4. IR spectra were obtained in KBr or as films
(CHCl3) on a Bruker Vector 22� IR spectrometer. All 1H, 13C and 2-D NMR experiments
were performed in CDCl3 on a Varian Unity 400� spectrometer equipped with a 5 mm
inverse detection pulse field gradient probe at 25� C at 400 MHz for 1H NMR and
100 MHz for 13C NMR. Chemical shifts were referenced to tetramethylsilane as an
internal standard.

3.2. Material plant

Aerial parts of P. brachystachyum, Nuttal were collected by Biol. Enrique Salazar Leyva
and Biol. Exp. Jesus Rivera Tapia, in October 2009 at Milpa Alta (19� 110 51 N and
099� 010 200 longitude) in México City. The voucher specimen was authenticated by
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Dr Miguel Chazaro and deposited at Metropolitano Ramon Riva y Nava Esparza

Herbarium (Universidad Autónoma Metropolitana-Iztapalapa, México, voucher number
70739).

3.3. Extraction and isolation

Aerial parts from P. brachystachyum (3.9 kg) were dried, powdered and extracted by
maceration at room temperature with acetone (3 L � 24 L, 48 h each) and concentrated to
dryness under vacuum to obtain 103.53 g of extract. Percolation of 60 g of this extract was
initiated with n-hexane 100%, followed by n-hexane-acetone gradient mixtures to render
117 fractions. On the basis of TLC, these fractions were pooled into four groups: A (1–17
fractions, 3.99 g), B (18–96 fractions, 12.97 g), C (97–108 fractions, 21.69 g) and D
(109–117 fractions, 8.87 g). A and C groups were resolved to pure compounds by means of
successive open CC (silica gel, 230–400 mesh), using a step gradient of n-hexane-acetone
100 : 0–90 : 10 or CH2Cl2 100% to CH2Cl2-acetone 90 : 10, as indicated. Group A was
divided in five subgroups: AI (50 mg, yielding fatty acids). AII [1.21 g residue, 4.5 cm
i.d. � 10 inch CC, n-hexane 100% to n-hexane-acetone 95 : 5, 112 fractions of 50 mL each
yielding squalene (527 mg, 0.88%) and �-sitosterol plus stigmasterol linoleates mixture
(478 mg, 0.80%)]. AIII [0.436 g residue, 2.0 cm i.d. � 10 inch CC, n-hexane 100% to
n-hexane-acetone 95 : 5, 30 fractions of 10 mL each yielding triacontanol (213 mg, 0.36%)
and �-sitosterol plus stigmasterol 80 : 20 mixture (138 mg, 0.23%)]. AIV [0.822 g, 3.0 cm
i.d. � 10 inch CC, n-hexane 100% to n-hexane-acetone 92 : 8, 64 fractions of 15 mL each,
yielding �-sitosterol plus stigmasterol 80:20 mixture (583 mg, 0.97%)]. AV [0.913 g, 3.0 cm
i.d. � 10 inch CC, n-hexane 100% to n-hexane-acetone 90 : 10, 85 fractions of 20 mL each,
yielding an �-amyrin and �-amyrin mixture (76 mg, 0.13%) and morolic acid (690 mg,
1.15%)]. �-Amyrin and �-amyrin were separated [76 mg residue, silica gel 230–400 mesh-
12% AgNO3, 1.0 cm i.d. � 10 inch CC, n-hexane 100% to n-hexane-acetone 95 : 5, 56
fractions of 10 mL each yielding �-amyrin (23 mg) and �-amyrin (3 mg)]. Group B
contained only 9.16 g (obtained by successive spontaneous precipitation as white powder,
15.26%) of pure morolic acid. Group C was divided into four subgroups: CI [0.139 g,
1.5 cm i.d. � 10 inch CC, CH2Cl2 100% to CH2Cl2-acetone 90 : 10, 42 fractions of 5 mL
each yielding squalene (76 mg, 0.13%), acacetin 7-methyl ether (58 mg, 0.097%)]. CII
[0.890 g, 3.0 cm i.d. � 10 inch CC, CH2Cl2 100% to CH2Cl2-acetone 90 : 10, 60 fractions of
50 mL each yielding acacetin (89 mg, 0.15%), lupeol (66 mg, 0.11%) and lupenone (79 mg,
0.13%)]. CIII [1.96 g, 5.0 cm i.d. � 10 inch CC, CH2Cl2 100% to CH2Cl2-acetone 90 : 10, 46
fractions of 20 mL each yielding betulinic acid and betulonic acid mixture (262 mg,
0.44%), betulin aldehyde (86 mg, 0.14%) and betulon aldehyde (110 mg, 0.18%)]. CIV
[16.98 g, 5.5 cm i.d. � 10 inch CC, CH2Cl2 100% to CH2Cl2-acetone 90 : 10, 166 fractions
 6 S. López-Martı´nez et al. Natural Product Research   135

of 100 mL each yielding oleanolic aldehyde (92 mg, 0.15%), oleanolic acid (420 mg,
0.70%), moronic acid (3.34 g, 5.6%) and morolic acid (10.80 g, 18.0%)]. Group D
contained only pure morolic acid obtained by spontaneous precipitation as white powder
(7.88 g, 13.13%). The structure of all these compounds was established via IR, 1H- and 13C
NMR data and comparison with those previously reported.

4. Conclusions
The hemiparasitic plant P. brachystachyum biosynthesises sterols (�-sitosteryl and
stigmasteryl linoleates, �-sitosterol, and stigmasterol), linear hydrocarbons (triacontanol
and squalene), oleanane-type triterpenes (�- and �-amyrin, oleanolic aldehyde, oleanolic
acid, moronic acid and morolic acid), lupane-type triterpenes (lupeol, lupenone, betulin
aldehyde, betulon aldehyde, betulinic acid and betulonic acid) and flavonoids (acacetin
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and acacetin 7-methyl ether). Morolic acid is its major constituents at 47.54 % yield, with
respect to dry total extract weight. This natural triterpene shows antihypertensive,
antidiabetic and anti HIV activities and the important amount isolated from this plant is
likely responsible for its medicinal use. P. brachystachyum is considered to be a nuisance
species, and this study demonstrated that it contains two major chemical compounds with
therapeutic potential.

Acknowledgements
This work was financially supported by CONACyT (Grant number 79584-Q). SLM thanks
ICYTDF for a postdoctoral fellowship. We are grateful to Biol. Enrique Salazar, Dr. Diana
Gabriela Vargas, Ing. Victoria Labastida, and T.C. Marı́a Medina Pastor for technical assistance.

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