Antibacterial activity of Helichrysum pedunculatum callus cultures

S. Ali'. J. Bol. 1999 . 64(5) 3 12- 313

312

Short Communications

Antibacterial activity of Helicltrysum pedunculatum callus cultures F. Dilika and J.J.M. Meyer* Department of Botany, University of Pretoria, Pretoria , 0002 Republic of South Africa ReC:(!ll 'ec/ 2- Apri1 199S: rt!\'ised l OJune / 998

Hellchrysum pedunculatum (Asteraceae) is used by the Xhosas of the Transke l ln South A frica to dress wounds after circumcis ion. As a verification of its folkloric use, the plant extract from this herb was earlier investigated for its an tibacterial activity. A callus cu lture of th is species was estab lished in half strength Murashige and Skoog

medium enriched with naphthalene acetic acid and kinetin. The homogenised dich lorometh ane extract of the ca llus was eva luated for its antibacte rial activity by direct bioautography on T LC The extract inhibited the growth of the Gram-positive bacteria, Bacillus cereus, B. pumilus, B. subtilis and Staphylococcus aureus as well as the Gram-negative bacterium , Serratia marcescens Keywords : Antibacteria l; callus; Helichrysum pedunculatum; tissue cultu re; traditiona l medicine. "To

whom

corre spondence

shou ld

be

addressed

(Email-

[email protected])

The antimicrobial properties ofa num ber of flel/cluJ'sulII (Asteraceae) species have bee n descri bed (Dekk er e/ a/. 1983; Boily & Vall Puyve lde 1986: T omas-Barbe ran el al. 1990; RiDs & Villar 199 1; Meyer & Afolayan 1995; Meyer & Dilika 1996). A South A fr ican spec ies. I fdiclu)'slIl1I ollr c ollilen.'i showed an tibacteri al activity against ,Iiicl'OCOCCliS kris/illoe, Bacillus cereus, B. pumUlls and S'faplrylococclIs (ll/l'eus Uvfey er & A fo layan 1995), Although work has been done on the anti m icro bia l aCli vity of liI.!lic/1I:l'slIlIl species, very little has been published on call us inductio n and the testing of these c ul tures fo r antibacteria l acti vity. Leeuwner and Meye r ( 1995) induced callus from H. aureOllilf!/I.\' and showed that it was act ive against /J suNilis, S. (///I'f!lIS, A:lebsiella pnelllJloniae and Escherichia coli lIelic!lIysul/1 pedllnc1IIalum Hil liard and Burrt has been repo rted in folklo re to have medicina l va lue. The se include its abil ity to curc stomach ai lments and its ant i- inflam matory activity (Watt & Breyer- Brandwijk 1962; Hilliard 1983; Bolofo & Jo hn son 1988). The pl ant is used by Xhosas in Trans kci (So uth Afric a) . to dress wounds especially after ci rc umcision, T he clai med medic inal pro perty of the plan t was su bstantiated by l'vl ey er and Dil ika (1996) who reported the antibacterial activity ofI he ext ract agai nst 8, ,'ercw', B. sublilis, S , pl/milu.\', ,\/. kl'islinoe. 5; aure llS, El1Ierohacfer cloacae an d ..\'errafiolllarCf!.H:ens. In this paper. we describe the establishment of H ped1l11cula1/111/ callus cultures a nd present the resu lts of their antibacter ial propert ies against ten bacterial species. Callus ind uctio n was ach ieved by surface sterili si ng you ng !eaves frol11 H p edutlculatlinT for 30 seconds in an aqueous solution of 0.35% sod ium hypochlor ite (NaDC l) followed by rinsin g 5 times in steri le distill ed water. Ste rile leaf material was then ex cised into 5 x 5 m m pi eces and placed on half strength M urashige and Skoog (1962) (MS) basal l11ed iu 111 (ICN Biomedica ls Inc .. UK) suppl emen ted wi th 3% sucrose, 1 O1gll naphthalene aceti c aci d (NAA) an d 0.05 mg/l kin eti n. The medium was solidified with 0.5% agar (Bio lab, SA). Homogenous cal li appeared within three week s (2 5°C. 24 h

day len gth; li ght intensity 35 ,5 ~t1n o l I1r~ S·I ) and were main tained by sub-cu lturi ng every six wecks Oll to fresh mcdium as described above but sol idifi ed wi th 0.3% agar. T he soft friabl e callus was spread cautious ly on the surface o f the mediulll. The spreading was necessary to secure a suffi c iently la rge area for secondary metabol ite production which is reported to be gen era ll y restricted to the upper ce ll layers (Dodds & Roberts 1986). Ca llus c ultures were mai ntained in light at 25°C and su b-cultured 011 fresh MS mediu lll every 6 weeks. The antibacterial bioassay was performed afte r five tra nsfers to fresh medium . Call us material was ag itated in cold water in order to remove the growth medium from the tissue wi t hout damaging the call us. dried and we ig hed (6,885g) . Metabo lites were extracted by homogen ising the cal lus in dichloroill ethane (CH~Cl 2 )' The homogenate was fi ltered and the sedi ment re~cxtracted by sti rring three ti m es in CH ~ C I: for 30 mi nutes eac h. The filtrate was concentrated to dryness under r~d ll ce d pressure , yield ing 39 mg residue. The sample was re-di sso lved in I Illi CHzC I ~ . The antibacte rial activ ity of the callus ex tract was tested against the following Gram-pos it ive bacteri al species: B (:(.!reus , R 1)l{lI1iitlS. 13. sub/ili.'i. ,\I. krislill{fe. S aurells and the Gram-n egative species: £ d onelle. E coli. }':. P"I1f.!1J10Iliae, P (Je/'ugillo,WI and 5; lI1a!'cescellS Eac h organism was maintain ed on nutrient agar (Bio lab, SA) and recovered for tcsting by growth in nutrien r broth (No.2. Biolab, SA) fo r 24 hours at 37°C except P lICrtlKi110SU which was harvested on ly aner 72 hours, T he anti bacterial activity of the ca llu s ext ract was tested by direct bioa utography on th in layer chromatog raphy (TLC) plates. The callu s ex tract (156 ~Lg from a 39 mg/ ml sol uti o n) was app lied to silica ge l 60 plates (Merck. Gerlllany), A ll extrac t of H. pedll11Clllutllm leav es (Meyer & Dilika 19(6) was also applied to the same TLC plate. T he plate was deve loped ill be nzcne:chl oroform (40: 60 v:v). It was observcd Linder UV light (2 54 and 366 nl11 ) after devc lo pment. len overn ight for the so lvent s to evaporate completely and sprayed w ith th e bacteri al suspensions, The bacterial sllspensions were obta ined as follows; 24 h (and 72 h P. lIt!rugi l1osa) o ld bacteria l cultu res in nu trie nt broth were centr ifuged at :3000 rpm for 20 m in. T he su pern atant was discarded and th e sed imented bacter ia re-sus pended in fresh Il utr ient broth to an absorbance of 0.8 4 at 560 11m (L und & Lyon, 1975). T he bac teri al suspensions were the n sprayed wit h a fine spray onto the TLC pl ates and Ihe pl ates dried for a few mi nutes unti l they appeared trans lusccnt. The plates were then incubated at 25 °C fo r 24 h (and 72 h fo r P aeruKil1o.<;a) in hum id cond itions whereafter they were spray ed with an aqueoll s sol uti on of 2.0 rng/ml p-iodonitrotet razol iulll violet a nd rei ncubated at 25°C for 3 to 72 h depending on the bacterial species. Any inhibition of bac teria l growth could clearly be seen as white spots on a red backgrou nd . More compo ll tlds were produced by the plant than the call us culture when observed 011 TLC plates, Thi s confirms the fi nd ings by Das and Law (1990), Thorpe ( 1990) and M issa leva "' a/. ( 1993), that secondary metabolites in a source plant Illay not be present in its cal lus . £. coli, r. aerllginosa, K. plleumolliae, i\I kristillae and F. cloacae showed res istance aga inst all the compounds of the callus extract. Certain compounds in the callus extract were, however , active aga inst B. cerellS, B pUl1/ilu.\'. /j subfilis. S (IIl/'ellS and S m(ll'cesccl1s. When the plant extract was tested wi th the agar diluti on method (Meyer & Dilika 1996), all the above mentioned G ram-positi ve bacteria were inhib ited as we ll as the Gram -n egative bacteria. 5; /J/ urcescens. E cloacae and AI. kristinae. Th e higher activ ity found in the agar dilutio n mcth od is probably due to the e ffect of the com bi ned antibacterial compounds in the extract when testcd ill Petri-dishes rath er than indi vid ua l com pounds be ing tested after be ing ch ro matographed on a TLC plate.

JIJ A Ithough not all the compounds produced by the plant were

found in the callu s extract, both extracts contained compounds that had more activity on Gram-positive bacteria than on Gram-Ilcgi1tive ones. T hese results are consistent with other st udies which also showed that pl ant extracts inhibit Gram-posi tive bacteria more than Gram-negative olles (Grosvenor el al. 1995; Meyer & Dilika 19961. Acknowledgements

S.A rr. .!. But. 1998. 64(5) 3 Il-3 1-l

In vitro culture of Mondia whitei (Periplocaceae), a threatened Zulu medicinal plant SA McCartan· and N.R. Crouch 1 "Durban Parks Department, Mlcropropagatlon Unit, 70 St. Thomas Road . Durban. 4001 Republic of Soulh Africa

We wish to thank the Foundation for Research Development of South Africa 101' thei r financial su pport.

' Ethnobotany Unit, National Botanica l Institute, clo Natal HerbarIum , Botanic Gardens Road , Durban, 4001 Republic of South Africa

References

Received II June 1998: revised 6 Augllsl/99S

BOILY . Y. & VAN PUYVELDE. L. 1986. Screening nf ml.!dicinal plants of Rwanda (Central Ali'ica) fu r anti microbial activity. J. EtllI1opharmam/. I (J: 1- 13 . B01.0FO. R.N . & JOHNSON. C. T. 1988. The identification of ' isicakathl' and liS lllcuicinai usc in Transkci. BOlhalia 18: 128- 130. DAS. N./'. &. LA W. K .II. I ()I}O . ProduCiion studies on Ilavonoiu:>. In : Fla"ounids in biology and medicinl.! III: CUO'l!llt issues in Ilavonoid rcsean:h, cd. NY I>as. pp . 39~7. NlltiollJi Un iversity of Singllpore. Singaporc. IlI ,KKE R. T.G .. fOIIR IE. T.G .. SNYCKERS. 1'.0. & VAN DER SC HYF. C.J. 1983. Studies llfSouth African medicinal plants. Pan 2. Cacspitin. a nc\v ph iomgluci no I dl.!rivativ~ with antim icrobial properties li'om flehdll:)'SUI1I Cl/e.ljJlIllllllII. SA. J Chem . 36 (4): 114-116. DODDS. J. I L &. ROBERTS. L.W. 1986. Experillll.!llts in PI;.uH TiSSlJe Cullure. 2nu cU. Cambridge tJnivcfSlIy Press. Cambridgc. GROSVENOR.I'.W .. SUPRJONO, A.S . &. GRA y, D.O. 1995. Medicinal plants [hIm Riau Province. Sumatra, Indonesia. Part 2: Antibacterial and antifung,,1act ivity. J. F:tIlllvpharlllllcol. 45: 97- 111. HILLIARD, O.M. 1983. Askr
II I. jI.'IEYER. J.J .M. & DILIKA, r 1996. Antibactl.!rial activit)' of Neli1..'111'):1'11111 peduncl/hllum llscd in circu mcis ion rites. J. Etlmoplwrlllacol. 5:1: 51 - 5-1. MURASHIGE. T. & SKOOG. F. 1962. A revised medium fIX rapid gnmlh and bioassays wilh tobacco tissue cultures. Phsiol. Plant. 15. -173. R[OS. M .e. & VILLAR , A 1991. ISl)lation <.md idclltilicatioll of the anti-

hacteri'll 1.,'tlJllpounus !l' om HelicJlI')!SIIIII slOec:has. J. Etlllloplmrll/acol. 33: 51 - 55. TIIOI{PE. T.A. 1990. Till! CUlTcnt statu:> ofphmt tissllc culture. In: Plant Ti ssue Culture: Appli cat ion and limitat ions, l!d. S.S. Bhoj wani. Elscvier Seiene!.! I)uhl ishers, Amsterdam. TOMAS-nARBERAN. FA. INIESTA-SANMARTIN. E., TOMAS-1.0RENTE. r. & RUMI3ERO. A. 1990. Antimicrobial phe!lolic compounds from three Spanish HelicJlI:vsul/I species. Phyfochem. 2(): 1093- 1095. WATT. .1M. & BREYER-BRANDWJJK. M.G. 1962. The medidnal and !lI.liso]HlllS plants of S(Jl lthem Alri ca. E.S. Livinsg.<;tnnc Ltd, Euinburgh.

Smgle-node explants of Mondia whitei (Hook.f.) Skeels derived from in vitro cultured seedl ings were used to produce rooted plantleIs on Ihe medium of Murashige and Skoog (1962) supplemenled with 1 mgl"1 SA, both in the absence and presence of cha rcoa l, and solidified with 0.3% w/v Gelrite . Eighty five percent of the plants were successfully hardened off under a 20/4 h lighUdark photoperiod and conditions of 80-100% humidity. M. white; is a highly prized and consequently over-exploited Zulu medicinal plant which is destructively harvested for its strongly aromatic roots. These are used for both their medicinal and food spice attributes. This micropropagation protocol allows for ca. 2000 plantlels to be produced from a single seed following 7 to 8 subcultures at 4 to 6 week intervals. Keywords : conservation, micropropagation, muthi, tissue culture , Zulu medicinal plant.

Mondia

whitei,

'To whom correspondence should be addressed. Present address: Department of Botany. University of Natal, P.Bag X01 , Scottsville. Pietermaritzburg , 3209 Republic of South Africa.

'\/rmdia while; ( Hook.f.) Skeels is the more widespread of the two species comprising the genus MUI/dia Skeels (Periplocaceae), both of w hi ch are li an as endcm ic to SOllth-Central and East Africa (Bullock 1961). At the tim e of its di scovery and subsequent description by taxonomists the over-exploitation of Mot/dia by the Zulu was clearly evident (Hooker 1871). By 19 15, Medley-Wood already consi dered M. whitei to be one of th e first medicinal taxa likely to become locally extinct. The popularity in Natal of AI whilei a century ago was chronicled by Bryant ( 1909): 'Every nat ive fort unate enough to procure (so me) habitually carries about with him a supply ... ofthe root and chews the same whenever the digestion may seek relier. Although its conservation status was recentl y listed by Hilton-Tay lor ( 1996) as 'Vulnerable ' in KwaZulu-Natal, the global status was given as 'not threatened ' . A status re-evaluation may result following consi deration of a report (Bouquet 1970 in Neuwinger 19(4) w hich desc ribes co mparabl e un sListai nable exploitation in tropical Africa, particu larly in the Congo. In KwaZulu-Natal, MOlldia was once known widely frolll coastal and midlands forests ; it is currently co nsidered extinct in the wild to the sO lLth of the Tuge[a river, and in Z ul uland is largely restricted to protected coastal swamp forests. Its popularity may stem from the pleasant aromatic character and taste of the rootstoc k (Bryant 1909): fro III the roots an i somer of va nillin has been iso lated (Gou lding & Pelly 19 11). Besides acting as a tonic (Med ley-Wood & Evans 1899) and settling the stomach (I-looker 1871 ), Ihe recorded medicinal uses of the root have variously included the easing of flatulen ce (Gerstner 1941) abdo minal pains and constipation, even the treatment of bilharzia (Gelfand el al. 1985). When mixed with Tiliacol'o CI1l:vsoboll)Y1 Welw. the combination is reported to form a mild laxative as we ll as a chest remedy (De Ficalho 1947). From Zimbabwe northwards to East-Central