Journal of Ethnopharmacology 68 (1999) 267 – 274

www.elsevier.com/locate/jethpharm

Preliminary screening of some traditional zulu medicinal

plants for anti-inflammatory and anti-microbial activities
J. Lin a,*, A.R. Opoku a, M. Geheeb-Keller a, A.D. Hutchings b,
S.E. Terblanche, A. K. Jäger c, J. van Staden c
a
Department of Biochemistry and Microbiology, Uni6ersity of Zululand, P. bag X1001, KwaDlangezwa 3886, South Africa
b
Department of Botany, Uni6ersity of Zululand, P. bag X1001, KwaDlangezwa 3886, South Africa
c
Natal Uni6ersity Research Unit for Plant Growth and De6elopment, Uni6ersity of Natal-Pietermaritzburg, P. bag X01,
Scotts6ille 3209, South Africa
Received 20 May 1999; received in revised form 12 July 1999; accepted 20 July 1999

Abstract

Aqueous and methanolic extracts from different parts of nine traditional Zulu medicinal plants, of the Vitaceae
from KwaZulu-Natal, South Africa were evaluated for therapeutic potential as anti-inflammatory and anti-microbial
agents. Of the twenty-nine crude extracts assayed for prostaglandin synthesis inhibitors, only five methanolic extracts
of Cyphostemma natalitium-root, Rhoicissus digitata-leaf, R. rhomboidea-root, R. tomentosa-leaf/stem and R. triden-
tata-root showed significant inhibition of cyclo-oxygenase (COX-1). The extracts of R. digitata-leaf and of R.
rhomboidea-root exhibited the highest inhibition of prostaglandin synthesis with 53 and 56%, respectively. The results
suggest that Rhoicissus digitata leaves and of Rhoicissus rhomboidea roots may have the potential to be used as
anti-inflammatory agents. All the screened plant extracts showed some degrees of anti-microbial activity against
Gram-positive and Gram-negative microorganisms. The methanolic extracts of C. natalitium-stem and root, R.
rhomboidea-root, and R. tomentosa-leaf/stem, showed different anti-microbial activities against almost all micro-or-
ganisms tested. Generally, these plant extracts inhibited the Gram-positive micro-organisms more than the Gram-neg-
ative ones. Several plant extracts inhibited the growth of Candida albicans while only one plant extract showed
inhibitory activity against Saccharomyces cere6isiae. All the plant extracts which demonstrated good anti-inflamma-
tory activities also showed better inhibitory activity against Candida albicans. © 1999 Elsevier Science Ireland Ltd. All
rights reserved.

Keywords: Anti-inflammatory activity; Anti-microbial activity; Zulu; Medicinal plants; South Africa; Vitaceae

1. Introduction

South Africa has an abundance of medicinal

* Corresponding author. Tel.: + 27-0351-93911-2280; fax:
plants, used in the traditional treatment of various
+27-0351-93162. diseases on an empirical basis (Hutchings and van
E-mail address: jlin@pan.uzulu.ac.za (J. Lin) Staden, 1994; Hutchings et al., 1996; Jäger et al.,

0378-8741/99/$ - see front matter © 1999 Elsevier Science Ireland Ltd. All rights reserved.
PII: S 0 3 7 8 - 8 7 4 1 ( 9 9 ) 0 0 1 3 0 - 0
268 J. Lin et al. / Journal of Ethnopharmacology 68 (1999) 267–274
1996; Salie et al., 1996; McGaw et al., 1997). Zulu
medicinal plants are traded and used all over
southern Africa. Scientific investigation and infor-
mation of the therapeutic potential of the plant
material is limited. With the possibility of tradi-
tional medical practitioners being integrated into
the state health delivery system, there is an urgent
need to systematically evaluate plants used in
Zulu traditional medicine. Such research could
also lead to new drug discovery or advance the
use of indigenous herbal medicines for orthodox
treatment.
Species from the Vitaceae are widely used by
traditional healers in South Africa to ensure safe
delivery during pregnancy (Hutchings et al., 1996;
Table 1). Rhoicissus tridentata is used to feed
children when mothers are not home (Tyiso and
Bhat, 1998). Cissus quadrangularis is used to treat
patients with burns, gastrointestinal complaints,
backache, body-and febrile pain, swellings and
malaria. The above medicinal plants along
with Cyphostemma natalitium from the same fam-
ily are also used to treat cancer patients (Hutch-
ings et al., 1996; Neuwinger, 1996). Resvera-
trol derived from Vitis 6inifera which also
belongs to the Vitaceae possesses anti-inflamma-
tory and anti-mutagenous activities and is a po-
tential cancer chemopreventive agent (Jang et al.,
1997).
Opoku et al. (1999) demonstrated that several
aqueous extracts from Zulu medicinal plants of
the Vitaceae showed anti-proliferative activities:
up to 97% inhibition in vitro against HepG2 cells.
Since these medicinal plants are also used to treat
burns, swelling and malaria, one can expect that
they might possess anti-microbial and anti-
inflammatory activities as well. The search for
antimicrobial and anti-inflammatory agents is
largely concentrated on lower plants, fungi
and bacteria (Fabry et al., 1998). Less research
focuses on higher plants, although, plant com-
pounds such as berberine, emetine, quinine, and
sanguinarine have specialised uses (Van Wyk et
al., 1997).
This paper reports on preliminary screening for
the anti-inflammatory and anti-microbial activi-
ties of nine plant species from three different
genera of the Vitaceae.
2. Materials and methods
2.1. Plant material
All plant material were collected during Febru-
ary and March 1998. The freshly collected plant
parts of Cissus quandrangularis L., Cyphostemma
fla6iflorum (Sprague) Descoings, Cyphostemma
lanigerum (Harv.) Descoings ex Wild and Drum,
Cyphostemma natalitium (Szyszyl.) J.V.D. Merwe,
Cyphostemma sp., Rhoicissus digitata (L.F.) Gilg
and Brandt, Rhoicissus rhomboidea (E. Mey. ex
Harv.) Planch, Rhoicissus tomentosa (Lam.) Wild
and Drum, Rhoicissus tridentata (L.F.) Wild and
Drum, and Rhoicissus tridentata (L.F.) Wild and
Drum subsp. cuneifolia (Eckl. and Zeyh.) N.R.
Urton, were identified by A. Hutchings and confi-
rmed by the National Botanical Institute.
Voucher specimens are lodged in the Botany De-
partment at the University of Zululand. The ma-
terials were thoroughly washed and separated into
leaves, stems and roots which were dried in an
oven at 50°C for 48 h. The dried material was
powdered (mesh size 2 mm) and stored in sterile
brown glass bottles at 4°C until use.
2.2. Preparation of plant extracts
Dry plant material was extracted using distilled
water in a Soxhlet extractor for 24 h at a temper-
ature not exceeding the boiling point of the sol-
vent. For each gram of dry material 2 ml of
solvent were used. The aqueous extracts were
filtered through Whatman No. 1 filter paper and
concentrated in vacuo at 40°C using a Rotary
evaporator. The residues obtained were desig-
nated as aqueous extracts and stored in a freezer
at − 20°C until assayed. Methanolic (MeOH)
plant extracts were prepared by extracting 100 g
powdered plant material three times in MeOH
(40% w/v), on a shaker at 150 rpm for 24 h at
room temperature. The extracts were combined,
filtered and the filtrates taken to dryness in vacuo
at 40°C. The residues obtained were re-dissolved
in 100% dimethyl sulfoxide (DMSO) and stored in
sterile brown glass bottles in a freezer at − 20°C
until bioassayed.
 J. Lin et al. / Journal of Ethnopharmacology 68 (1999) 267–274
2.3. Anti-inflammatory acti6ity assay
Cyclooxygenase is a key enzyme in the prosta-
gandin biosynthetic pathway which is important in
the inflammatory processes. The ability to inhibit
the COX-1 activity can be used to evaluate the
anti-inflammatory activity of any given compound
(McGaw et al., 1997).
The COX-1 activity was measured using the
method of White and Glassman (1974) as
modified by Jäger et al. (1996). The anti-inflamma-
tory activity of the extracts was expressed as the
percentage of inhibition of prostaglandin synthesis
using a COX-1 assay. The amount of [14C]-labelled
prostaglandin synthesized was measured using a
scintillation counter after removing the unmetabo-
lized [14C]-arachidonic acid substrate by column
chromatography. The percent inhibition (% I) of
prostaglandin synthesis was calculated as follows:
% I = 1− DMP of sample
−
DMP of backround
DMP of blank −DMP of backround
×100
where DPM is the disintegrations min − 1.
In all the assays performed, an ethanol in-
domethacin standard solution (20 m mol l − 1 in the
assay) was assayed together with the samples (13.3
mg for each sample residue) to verify the sensitiv-
ity of the assay. All experiments were performed in
duplicate.
2.4. Anti-microbial assay
The disc diffusion method of Salie et al. (1996)
was used to evaluate anti-microbial activities using
a range of microorganisms. Sterile AA Discs
(Whatman, 6 mm) were impregnated with 5, 10,
and 20 ml of reconstituted crude extracts (1000 mg
ml − 1) and placed on the surface of Muller – Hilton
agar dispersion plates inoculated with microbes.
Each extract was tested in triplicate. Control discs
contained 10 ml pure DMSO (100%). Standard
antibiotics, penicillin G (10 IU disc − 1), tetracy-
cline (30 mg disc − 1), and chloramphenicol (30 mg
disc − 1) were used to eliminate variation between
plates. Agar plates containing bacteria and fungi
269
were incubated at 37°C for 24 h and 30°C for 48
h, respectively. Inhibition zones were recorded as
the diameter of growth-free zones, including the
diameter of the discs, in mm, at the end of the
incubation period.
3. Results and discussion
Nine plant species from three different genera of
the Vitaceae were selected because of usage in
medicinal treatments in South Africa (Table 1).
Successful isolation of botanical compounds from
plant material is largely dependent on the type of
solvent used in the extraction procedure. The
traditional healers use primarily water as the sol-
vent. We found that in this study the plant extracts
by methanol provide more consistent anti-
microbial activity compared to those extracted
by other solvents. Therefore two solvents,
water and methanol, were used for the plant
n extraction.
Of the twenty-nine crude extracts assayed for
prostaglandin synthesis inhibition, only the five
methanolic extracts of C. natalitium-root, R. digi-
tata-leaf, R. rhomboidea-root, R. tomentosa-leaf/
stem and R. tridentata-root showed significant
inhibitory activity against COX-1 compared to
89% inhibition by the indomethacin standard (Fig.
1). The extracts of Rhoicissus digitata-leaf and of
Rhoicissus rhomboidea-root exhibited the highest
inhibition of prostaglandin synthesis with 53 and
56%, respectively. None of the aqueous extracts
showed any significant anti-inflammatory activity
(B 30%). Among those extracts that possessed
higher anti-inflammatory activities, only R. triden-
tata has been used to treat pains, swellings and
cuts or wounds by the traditional healers. The
results suggest that R. digitata-leaf and of R.
rhomboidea-root might have the potential to be
used as anti-inflammatory agents as well.
The growth media seem to play an important
role in the determination of the anti-microbial
activity. None of the crude plant extracts showed
significant anti-microbial activity against the
growth of Staphylococcus aureus (penicillin and
erythromycin resistant strain) on Blood agar
plates. However, on Muller–Hilton agar, some
Table 1

270
Names of Zulu medicinal plants used in this study and their therapeutic uses

Sample number Botanical name Voucher specimen Zulu name Part used Claimed therapeutic uses Extract
number

1 Cissus quadrangularis 1 AH and MG-K Isinwasi Root Burns, wounds, gastrointestinal complaints, backache, body- and MeOH
febrile pain, malaria (Hutchings et al., 1996), cancer (Neuwinger,
1996)
2 1 AH and MG-K Isinwasi Root Aqueous
3 5 MG-K Isinwasi Stem MeOH
4 Cyphostemma fla6iflorum 2 AH and MG-K Idambiso Leaf/stem During pregnancy to ensure safe delivery (Hutchings et al., 1996) MeOH

J. Lin et al. / Journal of Ethnopharmacology 68 (1999) 267–274

5 2 AH and MG-K Idambiso Root MeOH
6 Cyphostemma lanigerum 3747 AH Idambiso Root Cancer, during pregnancy to ensure safe delivery, toothache MeOH
(Hutchings et al., 1996)
7 Cyphostemma natalitium 6 MG-K Idambiso Leaf/stem During pregnancy to ensure safe delivery (Hutchings et al., 1996) MeOH
8 2 MG-K Idambiso Leaf/stem MeOH
9 6 MG-K Idambiso Root Aqueous
10 6 MG-K Idambiso Root MeOH
30 6 MG-K Idambiso Leaf/stem Aqueous
11 Cyphostemma sp. 4 MG-K Idambiso Leaf During pregnancy to ensure a safe delivery (Hutchings et al., MeOH
1996)
12 4 MG-K Idambiso Stem MeOH
13 4 MG-K Idambiso Root Aqueous
14 4 MG-K Idambiso Root MeOH
15 Rhoicissus digitata 3 AH and MG-K Isinwasi Leaf During pregnancy to facilitate delivery (Hutchings et al., 1996) MeOH
16 3 AH and MG-K Isinwasi Root Aqueous
17 3 AH and MG-K Isinwasi Root MeOH
29 3 AH and MG-K Isinwasi Leaf Aqueous
18 Rhoicissus rhomboidea 5 AH and MG-K Isinwasi Leaf MeOH
19 5 AH and MG-K Isinwasi Stem MeOH
20 5 AH and MG-K Isinwasi Root MeOH
21 Rhoicissus tomentosa 4 AH and MG-K Isinwasi Leaf/stem Antihelmintics to calves, during pregnancy to ensure a safe deliv- MeOH
ery (Hutchings et al., 1996)
22 4 AH and MG-K Isinwasi Root Aqueous
23 4 AH and MG-K Isinwasi Root MeOH
24 Rhoicissus tridentata 3 MG-K Isinwasi Leaf/stem Abdominal pains and swellings, anti-emetics in children, broken MeOH
bones, cuts, epilepsy, infertility, menorrhagia, during pregnancy
to ensure a safe delivery, renal complaints, sprained ankles, stom-
ach ailments, and sores (Hutchings et al., 1996; Van Wyk et al.,
1997; Veale et al. 1992)
25 3 MG-K Isinwasi Root MeOH
26 Rhoicissus tridentata subsp. 2 MG-K Isinwasi Root Aqueous
Cuneifolia
27 2 MG-K Isinwasi Root MeOH
 J. Lin et al. / Journal of Ethnopharmacology 68 (1999) 267–274
extracts demonstrated an significant capability to
inhibit the growth of S. aureus. Similar results
were observed when potato dextrose and SS agars
were used to assay the inhibitory activity against
Candida albicans and Salmonella-Shigella spp. re-
spectively. Muller–Hilton agar was found to be
the best medium to explicate the anti-microbial
activity.
The anti-microbial activities of all active ex-
tracts were concentration-dependent (data not
shown). Table 2, Table 3 shows the results of the
anti-microbial assay (20 mg of crude extract per
disc) against a range of microorganisms. Most of
the plant extracts showed variable inhibitory ac-
tivities against different microorganisms. The
methanolic extracts of C. natalitium-stem/root, R.
rhomboidea-root, and R. tomentosa-leaf/stem,
showed different levels of anti-microbial activities
against almost all microorganisms tested. The
methanolic extracts of R. rhomboidea-root and C.
natalitium-root generally demonstrated the
highest inhibitory activity against different micro-
organisms. The methanolic extract of R. digitata-
root mainly inhibited Gram-positive micro-
organisms and some Gram-negative micro-organ-
isms such as Pseudomonas spp and A. faecalis.
Most of the extracts showed poor inhibitory ac-
tivity towards Salmonella and Shigella spp. None
of the plant extracts were capable of inhibiting E.
Fig. 1. The percentage of inhibition of prostaglandin biosynthesis by the plant extracts in the cyclo-oxygenase (COX-1) assay. 13.3
mg of each sample residue was used in each assay.
271
coli. Generally, these plant extracts inhibited the
Gram-positive micro-organisms better than the
Gram-negative ones. This is in agreement with
previous reports that plant extracts are more ac-
tive against Gram-positive bacteria than Gram-
negative bacteria (Vlietinck et al., 1995; Rabe and
van Staden, 1997).
Factors that seemed to affect the anti-microbial
activity of the plant extracts were the location
and/or the season in which the plant materials
were collected. The methanolic extracts of C. na-
talitium-root (numbers 7 and 8 in Table 1) were
collected at different locations and within a 3-
month-interval showed dramatic differences in
anti-microbial activities.
Anti-fungal activity was tested using Candida
albicans and Saccharomyces cere6isiae (Table 2,
Table 3). The MeOH extract of R. digitata-leaf
and R. rhomboidea-root exhibited the highest ac-
tivity against C. albicans. No significant inhibitory
activity of the same extracts were observed
against S. cere6isiae. The methanolic extract of
Cyphostemma fla6iflorum (leaf/stem) was the only
one showing decisive (+ + + ) activity against S.
cere6isiae. Interestingly, all the plant extracts
which demonstrated good anti-inflammatory ac-
tivity also showed high inhibitory activity against
Candida albicans.
 272
Table 2
Anti-microbial activity of crude plant extracts from members of the Vitaceaea

Extract/or- A. faecalis B. cereus B. coagu- B. mega- B. pumilus B. B. subtilis C. albicans E. aerogenes E. coli K. pneumo- M. lutens M. phlei M. smegma-
ganisms lance terium stearother- nia tis
mophilus

1 − + + − ++ + + − − − − ++ − −
2 − + − − − − − − − − − − − −

J. Lin et al. / Journal of Ethnopharmacology 68 (1999) 267–274

3 − + − − − − + − − − − − − −
4 − ++ − − − − − − − − − − − −
5 + ++ ++ + ++ ++ − + − − + +++ − −
6 ++ ++ ++ + ++++ + − + − − + +++ ++ −
7 +++ ++ +++ +++ ++++ + − +++ − − + +++ ++++ ++
8 + + + − + − − − − − − + − −
9 + + + − ++ + − + − − − + − +
10 ++++ +++ +++ ++++ ++++ +++ + +++ + − + ++++ ++++ ++
11 − − − − − − − ++ − − − + − ++
12 − − − − + − + − − − − − − −
13 − − − − − − − − − − − − − −
14 − ++ + − + + − ++ − − + − − −
15 +++ ++ ++ − +++ ++ ++ ++++ − − ++ +++ +++ ++
16 ++ ++ ++ − +++ ++ − ++ − − ++ + + +
17 ++++ +++ +++ +++ ++++ ++ − +++ − − ++ ++++ − ++
18 +++ + + − +++ + − − − − − +++ − +
19 + + + − + + + + − − + − − +
20 ++++ +++ +++ ++++ ++++ +++ ++++ ++++ + − ++++ ++++ ++ +++
21 ++++ +++ ++ +++ ++++ +++ ++ ++ − − +++ ++++ ++ −
22 + + − − + − − − − − − − − −
23 ++++ +++ + +++ ++ ++ − − − − + +++ − ++
24 − − − − − − − − − − − − −
25 ++++ ++ +++ − ++++ ++ − ++ − − ++ ++ − ++
26 − + − − + − − − − − − ++++ − −
27 ++ +++ ++++ − ++++ +++ − − − − + ++++ − ++++
29 − − − − − − − − − − − − − −
30 − − − − − − − − − − − − − −
C ++++ − − ++++ − ++++ − − ++++ ++++ ++++ + ++ +++
Te ++++ +++ ++++ ++++ ++++ ++++ ++++ − − ++++ ++++ ++++ − ++++
DMSO − − − − − − − − − − − − − −

a
+: inhibitory activity where +, 2–4 mm; ++, 5–7 mm; +++, 8–10 mm; and ++++, \10 mm excluding the diameter of the disc; −: no activity; C, Chloramphenicol, 30 mg disc−1 (Oxoid); Te, Tetracycline, 30
mg disc−1 (Oxoid).
Table 3
Anti-microbial activity of crude plant extracts from members of the Vitaceaea

Extract/or- P. mirabilis P. mor- P. 6ulgaris Ps. Ps. sy- S. cere- Salmonella S. ty- S. marces- S. boydii S. flexneri S. aureus S. epider- S. faecalis S. pyroge-
ganisms ganii solanacearu ringae 6isiae sp. phimurium cens mis nes
m

1 + − − + − − − +++ − + ++ ++ ++ − −
2 − − − − − − − +++ − − − − − − −

J. Lin et al. / Journal of Ethnopharmacology 68 (1999) 267–274

3 − − − − − − ++++ + − − − + + − −
4 − +++ − ++ − +++ +++ + − − − + + − −
5 ++ ++ − + + − +++ + − − − ++ ++ + +
6 ++ ++ ++ +++ + − − − − − − ++ ++ ++ ++
7 ++ +++ + ++++ ++ − ++ − − − − ++ +++ + +++
8 + +++ + − + − − − − − − + − − +
9 + +++ + − − − − − − +++ − + − − +
10 +++ ++ ++ ++++ ++ − + ++ + ++ ++ ++ ++++ ++ +++
11 − − − − − − − ++ − +++ − − + − −
12 − − − − − − − + − +++ − − − − −
13 − − − − − − − − − − − − − − −
14 + − − − + − − ++ − − − + − − −
15 ++ + + ++ ++ − − ++ − ++++ − ++ +++ ++ ++
16 ++ + + + + − ++ ++ − − − + ++ − +
17 ++ − + +++ +++ − − − − − − ++ +++ + ++
18 − ++ − + + − − − − +++ − + ++ +
19 + ++++ ++ + − − ++++ + − +++ − + − − −
20 ++++ +++ ++ ++ ++ − +++ + + ++ − +++ +++ ++ ++++
21 +++ +++ + ++ + − + +++ ++ ++ − +++ +++ ++ +++
22 + − − − − − − ++ − − − + +++ − −
23 + +++ − +++ +++ − − ++ − − − ++ ++ ++ +
24 − − − − − − − + − − − − − − −
25 ++ ++ ++ ++ ++ − + − − ++ − ++ +++ ++ ++
26 + − − − + − +++ +++ − − − ++ + − +
27 ++ ++ + +++ +++ − − − ++ − − ++ +++ ++ +++
29 − ++ − − − − − − − − − − − − −
30 − ++ − + − − − − − − − − − − −
C ++++ − ++++ ++++ − − ++++ ++++ ++++ ++ + ++++ − ++++ ++++
Te ++++ + + + ++++ − ++++ − + − − ++++ − ++++ +
DMSO − − − − − − − − − − − − − − −

a
+: inhibitory activity where +, 2–4 mm; ++, 5–7 mm; +++, 8–10 mm; and ++++, \10 mm excluding the diameter of the disc; −: no activity; C, Chloramphenicol, 30 mg disc−1 (Oxoid); Te, Tetracycline, 30
mg disc−1 (Oxoid).

273
274 J. Lin et al. / Journal of Ethnopharmacology 68 (1999) 267–274
The results obtained demonstrate that many of
the Vitaceae plants screened in this study possess
potential anti-inflammatory and anti-microbial
activities. The usage of these plants by traditional
healers in the treatment of these ailments is there-
fore supported by these findings. Bioactivity
guided fractionation from highly active plants for
the isolation of inhibitory compounds is currently
underway. The isolation of new and effective anti-
inflammatory and anti-microbial compounds is
important both for potential drug development
and for the validation of the use of Zulu medici-
nal plants by traditional healers.
Acknowledgements
Funding for this project was provided by Uni-
versity of Zululand Research Fund, National Re-
search Foundation and University of Natal
Research Fund. Mr van der Linde from Owen
Sithole Agricultural College and Mr Wiggly from
the Twinstream Project provided some plant
material.
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