Phytomedicine, Vol. 5(3), pp.

209-214
Gustav Fischer Verlag 1998 _
I~~~~!~mml ..

Antibacterial and antifungal activities of

neocryptolepine, biscryptolepine and cryptoquindoline,
alkaloids isolated from Crypto/epis sanguino/enta
K. Cimanga', T. De Bruyne", L. Pieters', J. Totte ', L. Tona-, K. Kambu'', D. Vanden Berghe'
and A. J. Vlietinck 1
lDepartment of Pharmaceutical Sciences, University of Antwerp (UIA), Antwerp, Belgium
2Faculty of Pharmacy, University of Kinshasa, Kinshasa, Democratic Republic of Congo

Summary

From the 80% EtOH extract of Cryptolepis sanguinolenta (Lindl.) Schlechter (Periplocaeae) root bark,
a cryptolepine isomer named neocryptolepine, and two dimeric alkaloids named biscryptolepine and
cryptoquindoline were isolated. These compounds were tested for their putative antibacterial and anti-
fungal activities. Results have indicated that neocryptolepine showed an antibacterial activity against
Gram-positive bacteria (MIC <100 ug/ml), but was less acive against Gram-negative bacteria. It also in-
hibited the growth of the yeast C. albicans. Biscryptolepine exhibited only an activity against some
Gram-positive bacteria (MIC = 62.5 or 31 ug/ml) while cryptoquindoline did not shown an activity
against all selected microorganisms. The antibacterial activity of neocryptolepine and biscryptolepine is
bacteriostatic rather than bactericidal. No antifungal activity could be observed for all alkaloids in our
test system at the highest test concentration of 100 ug/ml,

Key words: Cryptolepis sanguinolenta, alkaloids, neocryptolepine, biscryptolepine, cryptoquindoline,

antibacterial and antifungal activities.

Introduction
Cryptolepis sanguinolenta (Lindl.) Schlechter (Peri-
plocaceae) is a climbing liana growing in some African
countries. An aqueous macerate or decoction of the
root bark or root is used in traditional medicine for the
treatment of fever including malaria, rheumatism,
amoebiasis and intestinal disorders (Kerharo and Ad-
am, 1974; Sofowora, 1982; Boye and Oku-Ampofo,
1990).
Cryptolepine (5-methyl-5H-indolo-[3,2-b]-quindo-
line) is the major alkaloid isolated from C. sanguino-
lenta collected from different African countries (Gellert
et al., 1951; Dwuma-Badu et al., 1978; Dwuma-Badu,
1987; Ablordeppey et al., 1990; Cimanga et al., 1991;
Tackie et al., 1991; Paulo et al., 1994b). In 1980s, the
variety of pharmacological effects of cryptolepine was
studied in detail (Bamgbose and Noamesi, 1981; Noa-
mesi and Bambose, 1980, 1983a, b, 1982, 1984; Oye-
kan et al., 1988; Oyekan and Okafor, 1989). More re-
cently, its antimicrobial activity on a large group of mi-
croorganisms including Gram-positive, Gram-negative
bacteria, yeasts and fungi (Cimanga et al., 1991; Paulo
et al., 1994a, b; Sawer et al., 1995), and its antimalar-
ial activity in vitro and in vivo (Noamesi et al., 1991;
Kirby et al., 1995; Wright et al., 1996; Grellier et al.,
1996; Cimanga et al., 1997) were reported.
In this decade, the root or root bark of C. sanguino-
lenta collected in different African regions was subject-
ed to phytochemical studies by several groups. This has
resulted in the isolation and structure elucidation of
different minor alkaloids (Tackie et al., 1993; Paulo et
al., 1994a; Crouch et al., 1995; Pousset et al., 1995; Ci-
manga et al., 1996a, b,; Sharaf et et al., 1995a, b, Sha-
raf et al., 1996a, b). The antibacterial (Paulo et al.,
1994b; Cimanga et al., 1996b), anticomplementary
210 K. Cimanga et al.
(Cimanga et al., 1996b) and antiplasmodial (Cimanga
et al., 1997) activities of minor alkaloids quindoline,
cryptoheptine and Ll-hydroxycryprolepine have been
investigated. It was also reported that isocryptolepine
exhibited an antiplasmodial activity (Grellier et al.,
1996).
Continuing the study of biological activities of other
minor compounds from this plant, the present paper
reports the antibacterial and antifungal properties of
three C. sanguinolenta alkaloids: neocryptolepine, bi-
scryptolepine and cryptoquindoline.
Materials and Methods
Plant material
Yellow roots of Cryptolepis sanguinolenta (Lindl.)
Schlechter were collected in Kinshasa/Congo in Octo-
ber 1989. The plant was identified by Mr Breyne of the
Institut National d'Etudes et de Recherches en Agrono-
mie (INERA) of the University of Kinshasa where a
voucher specimen has been deposited (INERA
892174).
Isolation and purification of neocryptolepine 1, bi-
scryptolepine 2 and cryptoquindoline 3
Powdered dried root of C. sanguinolenta (1.10 kg) was
exhaustively macerated and percolated with 80%
EtOH. The macerate and percolate were combined and
evaporated under reduced pressure to yield a dark red-
dish semi solid residue (88.93 g) and denoted F1. An
amount of 40 g was dissolved in 2x200 ml of distilled
water and filtered. The filtrate was exhaustively ex-
tracted with CHCI 3 The CHCl 3 and the aqueous layers
were separated and treated as described above to yield
the corresponding dried residues Fll = 13.74 g and
F12 = 24.13 g.
13.5 g of Fll was dissolved in a mixture of Me-
OH:H20 9:1 (2x200 m!) and filtered. The filtrate was
extracted with petroleum ether 40-65 (P.E.) and evap-
orated to yield a residue denoted Fll-1 (4.01 g). The
remaining aqueous MeOH phase was also evaporated
to dryness to give a dry residue Fll-2 (8.92 g). The res-
idue Fll-2 was dissolved in 10 ml MeOH and submit-
ted to column chromatography (CC) on silica gel
Merck (230-400 Mesh) (2x60 cm) eluted with a mix-
ture of CHCI 3:MeOH of increasing polarity. Several
fractions of 10 ml were collected and analysed by TLC
on silica gel 60F 254 Merck (layer thickness 0.2 mm) in
CHCI 3: MeOH 9:1 (solvent system 1) or in EtOAc:Iso-
PrOH:NH3 16:3:1 (solvent system 2). The spots were
detected under UV (254 and 366 nm) and after spray-
ing with Dragendorff's reagent. Similar fractions were
combined into three subfractions A, Band C according
to their chromatographic pattern.
Subfraction B was evaporated to dryness and the res-
idue (0.484 g) was dissolved in CHCl 3 (100 ml) and ex-
tracted with 1% HCl until Mayer's test was negative.
The acid fraction was basified with NH 3 25% (pH
9-10) and extracted exhaustively with CHCI 3 The
CHCl 31ayer was washed with distilled water and dried
on anhydrous Na 2S04 overnight in vacuo to give 0.213
g of a yellow residue after filtration and evaporation.
The residue was further submitted to CC on silica gel
Merck (230-400 mesh) (1.5 x 60 ern). The elution with
CHCl3 (400 ml) gave several fractions that were ana-
lysed by TLC as described above in solvent system 1.
Fractions with positive Dragendorff's test were com-
bined in one fraction according to their chromato-
graphic pattern. A yellowish compound with positive
Dragendorff's test was separated and isolated by prep-
arative TLC on silica gel (layer thickness 1 mm) in sol-
vent system 1: compound 1: yellowish, Rf = 0.92 (TLC
on silica gel, CHCI 3:MeOH 3:1), 17.3 mg (0.0035%
w/w from dried plant material or 0.13% from dried
chloroformic fraction F11).
Compounds 2 and 3 were isolated from the total al-
kaloid fraction obtained by the acid/base extraction
procedure for alkaloids as follows: 5 g of the total alka-
loid fraction were submitted to column chromatogra-
phy on active neutral aluminium oxide (AI20 3 ) Merck
(70-230 mesh) (2 x 120 em) in CHCI3:P.E. 2:1. The col-
umn was successively eluted with CHCI 3:P.E. 2:1 (1000
rnl), CHCI 3:MeOH 9:1 (1000 ml) and MeOH (500
ml). Several fractions of 10 ml were collected and ana-
lyzed by TLC (Merck, layer thickness 0.2 mm) using
'CHCI 3:MeOH:NH3 25% 8:2:0.5 as the mobile phase.
Spots were detected under UV (254 and 366 nm) and
after spraying with Dragendorff's reagent. Those giving
a positive test were combined according to their chro-
matographic pattern as described below:
- Elution with CHCI 3:P.E. 2:1 gave 3 major fractions
denoted Al (1.05 g), A2 (1.675 g) and A3 (0.472 g):
Fraction Al (1 g) was subjected to CC on silica gel
Merck (230-400 mesh) (2 x 60 ern) eluted with CHCl3
(250 mll, CHCI 3:MeOH 9:1 (500 ml) and CHCI 3:Me-
OH 7:3 (500 rnl). Several subfractions of 10 ml were
collected and analyzed as described above. Those elut-
ed with CHCI 3:MeOH 7:3 gave one subfraction re-
sponding positively to Dragendorff's reagent. This sub-
fraction (0.138 g) was further submitted to CC on sili-
ca gel Merck (230-400 mesh) (1 x60 ern) eluted with
CHCI 3:MeOH 4:1 (200 ml) giving 9 fractions of 10 ml
among which fractions 3 to 5 contained one chromato-
graphically pure compound with Dragendorff's test
positive, and denoted compound 2: 14.2 mg (0.28%
from total alkaloid extract or 0.0023% from dried
plant material), Rf = 0.095 (TLC on silica gel,
CHCI 3:MeOH:NH3 25% 8:2:0.5).
- Elution with CHCI 3:MeOH 9:1 resulted in 2 major
 Antibacterial and antifungal activities of neocryptolepine 211

fractions denoted A4 (0.367 g) and A5 (0.291 g) :The

residue from the evaporation of A5 (290.1 mg) was dis-
solved in 50 ml CHCI 3 The mixture was extracted ex-
haustively with 1% HC!. The acid layer was made al-
kaline with NH 3 25% (pH 9-10) and extracted with
90cc02
10

,;?'
8 ~
I
-"
oa
S
6a N Sa N 4a
lOb II

~
,;?'
I
,;?'
I
3
7 6 I 4
CHCl 3 (5 x 25 ml) until Mayer's test was negative. The CH3

CHCl 3layer was washed with water and dried with an- 1 Neocryptolepine
hydrous sodium sulfate overnight, followed by filtra-
tion and evaporation in vacuo to give 0.156 g of dried 2 Biscryptolepine
residue. This latter was further chromatographed on a
column packed with silica gel Merck (230-400 mesh)
(1 x 60 em). The elution with CHCI 3:MeOH:NH3 25%
8:2:0.5 gave 50 fractions of 10 ml analyzed by TLC on CH 3
silica gel (Merck, layer thickness 0.2 mm) in the same 6 I 4

eluant as the mobile phase. Spots were detected as de-
scribed above. Only fractions 20 to 34 contained one
chromatographically pure compound denoted com-
pound 3, 9.3 mg (0.18% from total alkaloid fraction or
0.0015 % from dried plant material), Rf = 0.95 (TLC
on silica gel, CHCl]:MeOH:NH] 25% 8:2:0.5).
Selected test microorganisms
The selected microorganisms included Gram-positive
bacteria: Staphylococcus aureus ATCC 6538, Strepto-
coccus pyogenes ATCC 12344, Bacillus cereus ATCC
14579, Mycobacterium fortuitum ATCC 6841, Gram-
negative bacteria: Escherichia coli ATCC 8739, Enter-
obacter cloacae ATCC 13047, Klebsiella pneumoniae
ATCC 13883, Proteus vulgaris ATCC 13315, Pseudo-
monas aeruginosa ATCC 15442 and Salmonella typhi-
murium ATCC 13311, the yeast Candida albicans
ATCC 102301, and fungi including Aspergillus fumig-
atus, Microsporum canis, Epidermophyton floccosum
and Trichophyton rubrum.
Culture medium
Bacteria and fungi were maintained on trypticase soy
broth (TSB) plates and Sabouraud dextrose agar plates
at 4 C respectively. Yeast were cultured in liquid Sa-
bouraud dextrose medium for 48 h at 24C. Test in-
ocula of fungi were prepared by harvesting mature
sporulating cultures in Sabouraud agar broth. For inoc-
ulation with fungi, homogenized mycelial cultures of
two weeks old were used.
Antibacterial and antifungal testing
The antimicrobial test of extracts and pure compounds
against bacteria and yeasts was performed by the mi-
crotiter plate dilution method whereas the microtiter
plate agar method was employed to test against fungi.
For this technique, an amount of extract or pure com-
pound was dissolved in DMSO, water and TSB (con-
centration of DMSO <1 %) to have a final concentra-
~
Sb SaN4~ 3
7 _ S I
2
ro
8~ e-, ~ ~
9 9a lOa 11 I
3 Cryptoquindoline 4 Cryptolepine
tion of 2 mg/m!. These stock solutions were repeatedly
diluted in twofold with TSB to obtain a series of con-
centrations of the test sample. An inoculum consisting
of about 10 6 microorganisms/ml TSB was incubated
overnight at 37C for 24 h. A 1/1000 dilution of this
suspension was prepared with the same medium, ex-
cept the suspension of M. fortuitum of which the dilu-
tion was 1/100. 100 I-tl of this dilution was brought into
the holes of a microtiter plate (11 bacteria on one plate,
one row containing one test microorganism). Thereaf-
ter, 100 ul of the test compounds in decreasing concen-
trations were added. Each vertical column contained
one dilution. The last column contained 100 I-tl TSB
and bacteria without test compounds, and was a con-
trol for the normal growth of bacteria. The plates were
incubated at 37C in humidified atmosphere for 24 h.
The inhibition of bacterial growth was evaluated by
comparing with normal bacterial growth in the control
holes prepared without test compounds. The minimum
inhibitory concentration (MIC) was determined as the
lowest concentration of the compound that completely
inhibited macroscopic growth of bacteria. To deter-
mine the minimum bactericidal concentration (MBC),
the two lowest concentrations which inhibited bacteri-
al growth were plated out on a nutrient agar and incu-
bated at 37C for 24 h. Results were evaluated by
comparing them with control holes containing bacteria
without test compounds (Vanden Berghe and Vlietinck,
1991).
Results and discussion
From the hydroalcoholic extract obtained from C. san-
guinolenta root bark, which exhibited an antibacterial
212 K. Cimanga et al.

Table 1. Minimum inhibitory (MIC) and minimum bactericidal (MBC) concentrations (ug/rnl) of fractions, alkaloids from C.
sanguinolenta root bark and reference antibiotic.
Fractions and Fl F2 1 2 3 4 5
compounds
Microorganisms MIC MBC MIC MBC MIC MBC MIC MBC MIC MBC MIC MBC MIC MBC
B. cereus 250 250 3.12 6.25 62 62 500 500 >100 >100 <7.8 15.75 <02 <0.2
C. albicans 500 >500 62.5 125 62.5 250 P >500 >100 >100 31.25 250 nt nt
E. coli 250 500 62.5 125 125 500 P >500 nt nt 62.5 500 3.12 3.12
Ent. cloacae 500 >500 250 500 250 500 >500 >500 nt nt 125 250 nt nt
K. pneumoniae >500 >500 >500 >500 250 >500 >500 >500 nt nt 500 500 nt nt
M. fortuitum 62.5 250 31 62.5 25 31 31 62 nt nt 6.25 12.5 nt nt
S. pyogenes <8 <8 <8 <8 <8 <8 <8 <8 nt nt <7.8 <7.8 <0.2 <02
P. vulgaris 62.5 250 31 62.5 31 62 >500 >500 nt nt 250 250 3.12 >25
P. aeruginosa >500 >500 >500 >500 250 500 >500 >500 >100 >100 250 250 >25 >25
S. typhimurium 250 250 125 250 125 250 >500 >500 nt nt 62.5 125 1.6 1.6
S. aureus 62.5 250 31 62.5 31 125 62 500 >100 >100 <7.8 125 6.25 >25
Legend: Fl - chloroform fraction from the partion of the 80% EtOH extract; F2 - total alkaloid fraction obtained by the ac-
id/base extraction procedure; 1 - neocryptolepine; 2 - biscryptolepine; 3 - cryptoquindoline; 4- cryptolepine; 5 - ampicilline
used as reference antibiotic; P - precipitation of compound in the culture medium; nt - not tested.

activity, some fractions were obtained through succes-
sive partition. Although some fractions showed an
antibacterial activity mainly against Gram-positive
bacteria, the CHCl 3 and the total alkaloid fractions
were selected for an extensive investigation because of
their pronounced antibacterial activity. In a previous
report, the bioassay-guided fractionation of these two
active fractions resulting in the isolation of quindoline,
cryptolepine and Il-hydroxycryptolepine was de-
scribed. These compounds exhibited to different degree
antibacterial properties against Gram-positive and
Gram-negative bacteria (Cimanga et aI., 1996a). In the
search of other antibacterial compounds from these
fractions, three alkaloids were isolated and identified
by spectroscopic methods (IR, UV, EI-MS, IH and
13C-NMR) as 5-methyl-5H-indolo-[2,3-b]-quindoline
named neocryptolepine 1, a cryptolepine isomer; and
two dimeric alkaloids, cryptolepin-ll-yl-cryptolepine
named biscryptolepine 2 and 11-(quindolin-l0N-yl)-
cryptolepine or cryptoquindoline 3, (Cimanga et aI.,
1996b).
The three alkaloids were submitted to the antibacte-
rial screening using a dilution method against a micro-
bial battery proposed by Vanden Berghe and Vlietinck
(1991). The antibacterial activity of cryptolepine is in-
cluded for a structure-activiy relationship. Results
shown in Table 1 indicated that compound 1 possesses
a wider antibacterial activity than compounds 2 and 3.
It exhibited an activity against B. cereus, M. [ortuitum,
P. vulgaris, S. aureus and the yeast C. albicans (MIC
<100 ug/rnl). It also inhibited the growth of some
Gram-negative bacteria such as K. pneumoniae, P. ae-
ruginosa and E. coli with MIC values from 250 to 125
ug/rnl. In general, the antibacterial effect of 1 is bacte-
riostastic rather than bactericidal. Compound 2
showed only an activity against M. [ortuitum, S. aure-
us (MIC <100 ug/ml) and weakly inhibited the growth
of B. cereus (MIC = 500 ug/rnl). The compound was
devoid of activity against all Gram-negative bacteria
selected and the yeast C. albicans (MIC >500 ug/ml).
Due to the aviable amount of compound 3, it was only
tested against B. cereus, P. aeruginosa, S. aureus and C.
albicans. Unfortunately, in our assay, the compound
was found inactive against these microorganisms at the
highest test concentration of 100 ug/rnl. However, in an
antimicrobial screening using Muller-Hinton's medi-
um, cryptoquindoline 3 exerted an antibacterial activ-
ity against S. dysenteriae, S. typhimuriurn, S. aureus, S.
faecalis (MIC ::;50 ug/rnl) and against V. cholerae (MIC
= 6.25 ug/rnl) (Paulo et aI., 1994a).
In spite of the strong antifungal activity observed for
the total alkaloid fraction against M. canis (MIC = 0.2
ug/rnl) , none of these compounds showed an activity
against E. [loccosum, T. rubrum and A. fumigatus at
the highest test concentration of 100 ug/rnl. In this as-
say, cryptolepine and its hydrochloride were also test-
ed. Cryptolepine HCI showed an interesting antifungal
activity against E. [loccosum, T. rubrum (MIC = 30
ug/rnl) and M. canis (MIC = 4 ug/rni]. The activity of
cryptolepine HCI against Microspsorum canis was
comparable to that of miconazole (MIC = 3.13 ug/ml)
used as a reference. Although the fungicidal activity of
cryptolepine base was reported against T. mentagroph-
ytes (MFC = 80 ug/ml) and S. cerevisiae (MFC = 40
ug/rnl) (Sawer et aI., 1995), the compound was found
inactive against all selected fungi in our assay system at
 Antibacterial and antifungal activities of neocryptolepine
the highest test concentration of 100 ug/rnl, This may
be due to the limited solubility of cryptolepine base.
The comparison of the antibacterial activity of cryp-
tolepine 4 to that of neocryptolepine 1, biscryptolepine
2 and cryptoquindoline 3 observed in our assay system
indicated that cryptole pine was more active than neo-
cryptolepine, except against P. vulgaris while the dim-
erisation as in 2 and 3 drastically diminished the anti-
bacterial activity.
In conclusion, although cryptolepine, the major alka-
loid can be considered as being responsible for the anti-
bacterial activity of the plant extracts, it appears from
this biological study that neocryptolepine contributes
to this biological activity.
Acknowledgements
K. Cimanaga was a recipient of a grant of the Algemeen
Bestuur voor Ontwikkelingssamenwerking (ABOS, Belgium).
T. De Bruyne is a post doctoral researcher of the Fund for Sci-
entific Research (FWO-Vlaanderen, Belgium). This work was
financially supported by the Flemish Government (Belgium)
(Concerted Action 92/94-09).
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Address
A. ] . Vlietinck, Department of Pharmaceutical Scienc-
es, University of Antwerp (UIA), Universiteitsplein 1,
B-2610 Antwerp, Belgium
Fax: (032) (03) 8202709