Journul of Elhnophurmacofogy, 35 (1992) 275-283 Elsevier Scientific Publishers Jreland Ltd.

27s

Screening for anti~icrubial activity of crude drug extracts and pure natural products from Mexican medicinal plants* Alejandra

Rojas”,

Lourdes

Hernandezb,

Rogelio

Pereda-Miranda’

and Rachel

Mata’

(Accepted September 24, 1991) Preliminary antimicrobiai screening against Candida afbbicansand selected Gram-positive and Gram-negative bacteria of methanol extracts prepared from eight Mexican medicinal plants, noted for their antiseptic properties, was conducted. The significant activity exhibited for extracts of Raiibida latipalearis, Teloxys graveolens, Dodonaea viscosa, HJ’ptis olbida, H. pectinato, H. suaveofens and H, verticiflora tends to support their traditional use as anti-infective agents. Only the extract of Hintonia /ati/7oru was inactive. The antimicrobial activities of 44 pure natural compounds and two derivatives were determined. Of these, only 23 compounds were effective in inhibiting the growth of the tested organisms (MIC 5 100 &ml). Key words: antimicrobial screening; anti-infective agents; folk medicine

The screening of plant extracts and natural products for antimicrobial activity has revealed the potential of higher plants as a source of new antiinfective agents (Mitscher et al., 1972, 1987). As part of a research program directed towards the isolation of active compounds from Mexican plants used in traditional medicine, this paper describes (i) the preliminary antimicrobia1 screening of eight crude drug extracts and (ii) the activities of 44 pure natural compounds isolated From medicinal plants. The plants used in this study are conveniently avaifabfe from the Mexican markets and have a reputation for their antiseptic properCorrespondence lo: R. Mata, Laboratorio de Fitoqimica, Departamento de Farmacia (DEPg), Division de Bioquimica y Farmacia, FacuJtad de Quimica, Universidad National Aut6noma de Mexico, 04510 D.F., Mexico. *Part XXIII: Chemical Studies on Mexican Plants Used in Traditional Medicine and Part IV: Chemical Studies on Mexican hlypris Species. Taken in part from the MS thesis of A. Rojas and L. Hernlindez.

ties. The selected species were Ra~ibida iu~~p~leur~s Richards (Asteraceae), Z”eloxysgr~~e~~e~s~ilid.) Weber (Chenopodiaceae), Hintopzia~ur~~#ra(Sesse et Mocifio ex DC.) Bullock (Rubiaceae) (Bye, 1986), Dodonaea viscosu (L.) Jacq. (Sapindaceae) (Martinez, 1989), Hyptis pectinata (L.) Poit. (Malan et al., 1988). H. ulbida HBK., U. SUQVCO/ens (L.) Poit. and H. verrjc~/i~r~Jacq. (Lamiaceae) (Martinez, 1989). Regarding the pure naturat compounds, 25 were isolated from the above plants and six were obtained from ~~~~~.~~~er~~g~~~ ~d~~~~~gensSchiede ex Schlecht. ffutianiaceae) (Estrada, 1985) and H@p@crafea excelsa {Hippo~ratea~ae~ HBK. (Pafacios et al., 1989), which also have fofkloric reputation as anti-infective agents. Finally, the remaining 12 compounds were isolated from three Rubiaceae species, namely Exostema caribaeum (Jacq.) Roem. et Schult. (Mata et al., 1987b; 1988b), E. mexicanurn Gray (Mata et al., 1990b) and Cigarrillu mexicana (Zucc. et Martius ex DC.) Aiella (Mata et al., 1988a) and from three Lamiaceae species, namely, Sulvia ~lboc~erul~

0378-~74ll92JSOS.~ 0 1992 Elsevier Scientific Publishers Ireland Ltd Printed and Published in Ireland

276

Lindl. (Pereda-Miranda et al., 1991), S. nicalsaniana Ramamoorthy (Pereda-Miranda and Delgado, 1986) and H@s ~~~~~~1~~~~Benth. (Pereda-Miranda et al, 1990), plants used for other therapeutic purposes. In addition, two synthetic compounds derived from other plant constituents were evaluated.

identification have already been described by us in the references included in Table 1. Preparation

of crude extracts

Materials and Methods

In each case, 100 g of the air-dried material, finely ground, was exhaustiveiy extracted by percolation with methanol. The extracts were evaporated under vacuum to a thick residue which was stored under nitrogen at 4°C until tested.

Plani material

Screening for ant~mj~robia~ activity

All plants used in this study were present in our laboratory because of initial interest in chemical studies. Aerial parts of H&t& vert~~jl~atu were purchased in the public market Central de Abaslos in Oaxaca City and Tetox??;sgraveo~ens in Mercado de Sonora in Mexico City. Stem bark of Hintonia latifrora and roots and leaves of Ratibida latipalearis were obtained from the Chihuahua City market. Leaves of Hyptis albida were bought from a native healer in Huetamo, State of Michoac&n. Aerial parts of Dodonaea viscosa were collected in JayacatlBn, State of Oaxaca. Finally, aerial parts of Hyptis pectinata and fY. suave&ens were collected in Xalapa, State of Veracruz. For each plant studied, voucher herbarium specimens were prepared and submitted for identi~~tion by a botanist at the Instituto de Biologia, UNAM. Vouchers of Ratibida latipalearis [Bye and

The crude extracts and pure compounds were evaluated for qualitative antimicrobial activity using the agar plate diffusion assay (Hufford et al., 1975; Rios et al., 1988). Determination of minimum inhibitory concentration (MIC) was accomplished by the two-fold serial dilution technique (Hufford et al., 1975). The following strains were used as test organisms: Staphy~oco~&~s aweus (ATCC 6538), Bacillus subtilis (ATCC 6633) Escherichia coli (ATCC 8739), Pseudomonas aeruginosa (ATCC 9027) and Candida aibicans (ATCC 1023 I ). Nutrient agar no. 1 (Merck) was used for bacterial cultures and Sabouraud agar (Merck) supplemented with glucose (4%) for yeast. Nutrient broth (Merck) and Sabouraud~glucose (2%) nutrient broth (Merck) were used for MIC determinations. Agar plates containing 1 ml (IO6 bacteria/ml) of an overnight broth culture were prepared. Holes having a diameter of 11 mm were made and filled with 100 11 of the test solution. The crude extracts were assayed as aqueous suspensions in 1% Tween-80 at a concentration of 20 mg of extract per ml. Pure compounds were tested at 1 mg/ml. The plates were then incubated at 37°C for 24 h, except for C afbicans which was incubated at 25°C. Streptomycin sulfate (Sigma) (I mglml) and nystatin (Sigma) (3 mgiml) were used as positive controls. The diameters of the resultant inhibition zones were measured. As each experiment was performed in triplicates, the means of the diameters for the inhibition zones were determined. FOF the quantitative antimicrobial assays, each test compound was dissolved in MeOH-HZ0 (1 mg/ml); 0.5 ml of this solution was then added to 4.5 ml of nutrient broth. Doubling serial dilutions

~~~~~~rt~y

16656),

Teloxys

gra~eo~~~~

{MC-89-l). Hyptis albida (M-8528),

H. pectinata (M-21853), H. suave&ens (M-21850), H. verticillata (MEXU-431167) and Hinton~a latijlora (Bye and tinares lfSf6) have been deposited in

the National Herbarium of Mexico (MEXU), Instituto de Biologia, UNAM, while reference specimens of ~udonaea viscosa (Cisneros 368) have been deposited in the Herbarium of the Department of Botany, School of Chemistry. Universidad Autonoma Benito Juarez de Oaxaca, Oaxaca, Mexico. Pure Compounds

The natural products used in this study were previously isolated from Mexican medicinal plants. The test compounds and their plant origin are listed in Table 1. The molecular structures of l-44 are shown in Figs. 1-6 and details on their

277 TABLE I NAME AND ORIGIN OF TEST COMPOUNDS Compound

Name

Origin

Reference”

1

5-O-B-D-Glucopyranosyl-7,3 ’ ,4 ‘-trihydroxy-4phenylcoumarin 5-O-(6’ ‘-Acetyl)-&o-galactopyranosyl3 ‘,4’-dihydroxy-7-methoxy-4-phenylcoumarin 5-O-(6’ ‘-Acetyl)-b-D-glucopyranosil-7,3,4’trihydroxy4phenylcoumarin 5-0-@-D-Glucopyranosyl-3’,4’-dihydroxy-7methoxy4phenylcoumarin 5-0-b-o-Galactopyranosyl-3’,4’-dihydroxy7-methoxy4phenylcoumarin 7,5,3’,4’-Tetrahydroxy4phenylcoumarin 3’,4’,5-Trihydroxy-7-methoxy-4-phenylcoumarin 7,4’,5’-Trihydroxy-4-phenyl-5,2’-oxidocoumarin 4,5’-Dihydroxy-7-methoxy4phenyl-5,2’oxidocoumarin Chrysin Pinostrobin Apigenin-7,4’-dimethyl ether Luteolin Luteolin ‘I-methyl ether Hispidulin Cirsimaritin Ermanin Kaempferol 3,7,4’-trimethyl ether 5,6,4’-Trihydroxy-3,7-dimethoxy-flavone Nevadensin A Gardenin B Pinocembrin Sakuranetin lsosakuranetin Cucurbitacin F Cucurbitacin F-25-acetate 23,24_Dihydrocucurbitacin F 23,24Dihydrocucurbitacin F-25-acetate 2-0-/3-o-Glucopyranosylcucurbitacin F-25-acetate 3-0-@-D-Glucopyranosyl-23,24-dihydrocucurbitacin F Arvenin I Canophilol Canophilic acid Canophilal Masticadienonic acid Isomasticadienonic acid 3-a-Hydroxymasticadienonic acid Hautriwaic acid Enf-I 5,16-Epoxy-3/3,8a-dihydroxy, 9oH-labda- I3( l6), ICdiene Sugiol I5-Hydroxy-7-oxo-abieta-8, I I, l3-triene Hyptolide

Hinronia latiflora

I

Hinronia Iarifora

I

Hinronia latiflora

I

2 3 4 5

10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 38 31 32 33 34 35 36 37 38 39 48 41 42

Himonia Iat$ora

I

Hintonia latijlora

I

_b _b Exostema Exostema

caribaeum caribaeum

Teloxys graveolens Teloxys graveolens Hypris albida Salvia nicolsoniana Hintonia latiflora Ratibida laripalearis Hypris albida Hyptis albida Hypris albida Dodonaea viscosa Hyptis albida Hyptis albida Teloxys graveolens Dodonaea viscosa Hypris albida Exosrema mexicanurn Exostema mexicanum Hinronia latiflora Hinronia latijlora Cigarrilla mexicana Hinronia larijlora

4 4 5 6

I 7 5 5 5 8 5 5 4 8 5 9 9

IO IO II I

Cigarrilla mexicana Hippocrates excelsa Hippocrates excelsa Hippocrates excelsa Amphypterygium adsrringens Amphypterygium adstringens Amphyplerygium adstringens Dodonaea viscosa Dodonaea viscosa

II I2 I2 I2 I3 I3 I3 8 8

Salvia albocaerulea Salvia albocaerulea Hyptis pectinala

I4 I4 I5

27X

TABLE I (Conrim&) NAME AND ORIGIN OF TEST COMPOUNDS Compound

Name

Origin

Reference”

43

4-Deacetoxy-IO-epiolguine 6-(5’,6’-Diacetyloxy-I ‘,2’-dihydroxy-3heptenyl)-5,6-dihydro-2H-pyran-2-one 6-(5 ‘,6’-Diacetyloxy-I ‘hydroxy-2’-methoxy3-heptenyl)-5,6-di-hydro-2H-pyran-2-one 6-( I ‘,2’,5’,6’-Tetracetyloxy-3-heptenyl)5,6-dihydro-2H-pyran-2-one

Hypris oblongr@olia Hypiis oblongryolia

I5 I5

Hyplis oblongijolia

15

Hypris oblongifolia

I5

44

45 46

“References: (I) Mata et al., 199Oa;(2) Mata et al., 1987b;(3) Mata et al., 1988b; (4) Mata et al., l987a; (5) Pereda-Miranda and Delgado, 1990; (6) Pereda-Miranda and Delgado, 1986; (7) Rojas et al., 1991; (8) Mata et al., 1991; (9) Mata et al., 199Ob;(IO) Reguero et al., 1987; (I I) Mata et al., 1988a; (12) Calzada et al., 1991; (13) Navarrete et al., 1989; (14) Per~a-Miranda et al., 1991; (15) Pereda-Miranda et al., 1990a. bDerived from other plant compound.

were aseptically prepared from this broth with concentrations ranging from 100-0.2 &ml. Each dilution was inoculated with 10 ~1 of bacteria to a final concentration of 106/ml. After overnight incubation, the MIC was determined. Streptomycin

*I

40 R4

R2O

on

10 R,=Rz=R3=Rs=RS=Rg=H;11 R,=RZ=Rs =R,=R,=H,R,=Me;12 R,=OMe,R1=Rj=Rq=Rr, = H, R5 = Me; 13 R, = R, = OH, R, = R4 = Rs = Re = H; OH. R, = R, = R, = H, RS = Me; 15 R, = 14 R,=R,= OH, R, = R, = R5 = R, = H, R, = OMe; 16 R, = OH, R, = R, = R, = H, R4 = OMe, R, = Me; 17 RI = R3 = OMe, R2 = R4 = R5 = R, = H; 18 R, = R3 = OMe, R, = R4 = R6

HO 1

R, = &o-glucose, R, = H; 2

Rt = 6-acetyl-P-D-galactose, R, = Me; 3 R, = 6-acetyl-P-D-glucose, R, = H; 4 R, = P-Dglucose, R, = Me; 5 R, = P-D-galactose, R, = Me; 6 R, = R,=H;7

*o

= H, R, = Me; 19

R, = R, = OH, R, = R, = H, Rj = OMe.

R, = Me; 20

R, = R4 = R, = OMe; R, = R, = Rs = H; 21 R1 = R, = R, = OMe; R, = R, = H; R, = Me.

R,=H;Rz=Me.

\o

0

P L

0

OI’’

*20

R = H:9

ON

I=

0

22 R, = R, = H; 23 R, = H. 1-Y

Rl

/

OH

R = Me

Fig. I. Structures of phenylcoumarins

aJr \o

I

HO

8

0

R, = OH, R, = Me; 24

Fig. 2. Structures of flavonoids

lo-24

R, = OMe,

279

Results and Discussion

those of the widely used antibiotics streptomycin and nystatin. As indicated in Table 2, only the extract of Hintonia latifora was inactive against all the test organisms. The extracts of Hyptispectinata and H. suaveolens were only active against the Grampositive bacteria. Teloxys graveolens showed marginal activity against Bacillus subtilis, Pseudomonas aeruginosa and C. albicans. However, the extracts of Ratibida latipalearis, Hyptis albida, H. verticillata and Dodonaea viscosa significantly inhibited the growth of all tested microorganisms at extract concentration of 20 mg/ml. It is important to point out that the remarkable inhibition exhibited by the extracts of R. latipalearis and H. verticillata against C. albicans deserves further screening for antifungal activity (Mitschner et al., 1972; Adeoye et al., 1986). The results of this preliminary screening, in particular for the extracts of R. latipalearis, H. albida, H. verticillata and D. viscosa, tend to support the use of these plants as anti-infective agents in folk medicine.

Antimicrobial

Antimicrobial

25

R, = a-OH,

R, = (Y-OH, B-H, R,

O-H, R, = R, = H; 26

= H, R, = AC; 27 R, = a-OH, /3-H, R, = R, = H, 23,24-dihydro; 28 R, = (Y-OH, 8-H. R, = H, R, = AC, R, = 23,24-dihydro; 29 R, = (Y-OH, /3-H, R, = fi-~-glucose, AC; 30 R, = ru-O-@-glucose, O-H, R, = R, = H, 23,24-dihydro; 23.24-dihydro.

31

Fig. 3. Structures

R,

= 0,

of cucurbitacins

R,

=

&D-ghcose,

R,

= H,

25-31.

and nystatin, prepared in the same manner, were used as positive controls. Two series of determinations were run for each organism.

screening of extracts

32

R = CH20H;33

Fig. 4. Structures

R = COOH;34 of triterpenoids

32-37.

R = CHO.

activity

of pure compounds

Of the 44 natural products and two derivatives tested, only 23 showed significant antimicrobial activity in the diffusion experiments and for these compounds further dilution studies were conducted. These results are summarized in Table 3. Most of the active products were among the flavonoids. As expected, the active compounds

Table 2 shows the antimicrobial activity against Candida albicans and selected Gram-positive and Gram-negative bacteria of the extracts prepared from eight Mexican medicinal plants, which have been used in treating infectious diseases. The activities are expressed by the diameter of the developed inhibition zones and compared with

35

R = 0, A’; 36

R = 0,A8’9’; 37

R = (Y-OH, 6-H; A’.

280

:: fl

I’

:

no-’

’

OH

Q

, I

.

40

38 Fig. 5. Structures

of diterpenoids

OAc

portant feature for activity. Also, a free hydroxyl group at C-7 seems to be a structural requirement for activity in the oxidocoumarin subgroup. Among the triterpenoids, in the cucurbitane series, only the curcurbitacin F-25 acetate (26) exhibited a good activity against P. aeruginosa. A comparative analysis of compounds 25-31 allows one to speculate that the free hydroxyl group at the C-2 and C-3 positions, the 25-acetate unit and the conjugated double bond between C-23 and C-24 are structural requirements for the observed effect. Regarding the friedelanes, the only active compound was canophilal (34), which also exhibited specific activity against P. aeruginosa. None of the evaluated tirucalanes displayed a noteworthy effect on microbial growth. Finally, the diterpenes (38-41) and the LXpyrones (42-46) only showed marginal activity against the Gram-positive bacteria. The relatively high proportion of active compounds present in the extracts indicates, as previously demonstrated by Mitscher et al. (1987),

OAc .

OAc

43

42 of a-pyrones

R, = H; R, = OH.

38-41.

possess a free hydroxyl group at C-7 of the basic skeleton (Mitscher et al., 1987) with the exception of 16. The cirsimaritin (16) and pinocembrin (22) showed the widest spectrum of activities and their presence, in relatively high yields, in H. uf~~~a (Pereda-Miranda et al., 1990b) and T. graveoiens (Camacho et al., 1991), respectively, could account for the antiseptic properties demonstrated in this study for the crude drug. The activity exhibited for chrysin (10) against P. aeruginosa and C. alb~cans is noticeable. In spite of the phenolic nature of most 4-phenylcoumarins, this type of natural compound has not been previously subjected to any antimicrobial screening. The results showed in Table 3 clearly indicate that these natural products do not exhibit a wide spectrum of antimicrobial activities. However, it is important to point out the selective activity of 5 and 8 against C. albicans in comparison with the lack of activity displayed by 4 and 9. It appears that in the case of compound 5, the presence of the galactosyl moiety is an im-

Fig. 6. Structures

R, = OH, R, = H; 41

42-46.

44

R,=R,=H;45

H

R,=H;R?=Me;46

R,=Rz=Ac.

281

TABLE

2

SCREENING

FOR ANTIMICROBIAL

Plant material

ACTIVITY

OF 8 CRUDE

DRUG

Part used”/ extract yield (1%)

Local uses

Local name

EXTRACTS Inhibition zone (mm) per microorganismb I

Teloxys graveolens

Epazote de zorillo

Hyptis albida

Salvia blanca

Hyptis pectinata

Hierba del burro Hierba del

Hyptis suaveolens

Dodonaea viscosa

burro Hierba martina Chapulizte

Hintonia latiflora

Copalchi

Standard’ Vehicle Control

-

Hyptis verticillata

PL (8.6)

Wound healing, headaches Stomach infections, anthelmintic, intestinal ailments Wound healing, anthelmintic Skin infections, lung congestion Wound healing,

Ratibida htipalearis

5

2

3 3

7

7

-

4

4

LF (13.6)

6

8

5

PL (7.3)

6

IO

-

LF (9.4)

6

9

-

-

LF (18.6)

6

IO

6

7

LF (10.9)

6

8

5

5

7 -

I2 -

7 _

9 -

SB (21.2)

-

-

“(LF) leaves; (PL) whole plant; (SB) stem bark. Yield = w/w in terms of dry starting material. bMicroorganism: (I) Staphylococcus aureus; (2) Bacillus subtilis; (3) Escherichia coli; (4) Pseudomonas albicans. cAntibiotic

TABLE

standards:

streptomycin

sulfate

for bacteria

and nystatin

aeruginosa;

(5) Candida

for C. albicans.

3

ANTIMICROBIAL ACTIVITY OF PURE COMPOUNDS TIONAL MEDICINE AS ANTISEPTIC AGENTS Compound

b

Zone of inhibition, S. aureus

mm (MIC, B. subtilis

E. coli

3

16

7 (31.25)

MEXICAN

MEDICINAL

C. albicans

7 (50) 9

(15) 7

(31.25)

(25)

II

8

3

(15) 3 3

(31.25)

3

6 (45)

-

PLANTS

P. aeruginosa

7 (12.5) 8

10

15

FROM

&ml)”

3 S

I7 20

5

9

PL (15.4)

skin infections Wound healing, headaches Wound healing, skin infections Antimalarial, stomach infections, wound infections

4

-

USED

IN TRADI-

282

Compoundh

Zone of iflhjbition.

mm (MIC,

pglml)”

3

21 22

I

24

(50) I (50)

26

4

8

I

(25) 3

(50) -.

9 (25) 9

34

(75) 2 3

37 38

_

6 (75)

39 40

4 6

10

41

(50) 6

(40) 9

(50)

(45) 5

42

(1W 6

43

(100) 44

6

45

f100) 7

_

flW 6

46 Streptomycin

7

(1@0) 12

7

9

(3.12)

(1.56)

(3.12)

(12.5) 9

-_

Nystatin

(6.25) -“Tube dilution

studies (MIC)

in the diffusion bTable

were conducted

for only those compounds

____.

considered

to have significant

inhibition

zones

( 3 5 mm)

experiments.

1 compounds

not included here were inactive in the diffusion

that the selection of a plant for research by reference to its use in traditional medicine may indeed be a useful criterion to identify new antiinfective agents.

studies.

(UNAM) for the provision of facilities to conduct some of the evaluations. This research was partiatly supported by Projects PADEP No. FQ-9125 and FQ-9127 from the Coordinacicin General de Estudios de Posgrado, UNAM.

Acknowledgements References The authors are indebted to Dr. Ramon Cruz Camarillo, Laboratorio de Enzimas Microbianas, Departamento de Mi~robiolog~a (ENCB), Intituto Politknico National and to Q.F.B. Marta Jimenez, Cepario de la Facultad de Qujmica

Adeoye,

-

A.U.,

_

Ctguntimetn,

KU.,

Clark,

C.D. it9861 3-~imethyial~y~~ndo~e: tifungal

metabolite

Nutuml

Proclucis 49, 534-537.

Bye. R.A.,

from

Monodora

Jr. 11986) Medicinal

A.M.

and H&ford,

An antibacterial tenuifolia.

and an-

Journul of

plants of the Sierra

Madre:

283

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