FLABELLIFERINS, STEROIDAL SAPONINS FROM PALMYRAH (BORASSUS FRUIT PULP 11. Preliminary investigations of effect on yeast and selected bacteria

I Abstract:, The isolation and partial characterization of four naturally occurring , '. .-, -/. ') steroidal saponins (flabelliferins) differing in their carbohydrate moiety and . , isolated from palmyrah fruit pulp was reported.previously. These were called flabelliferin F-I1 (a tetraglycoside), flabelliferin F, and % (triglycosides) and tlabelliferin F, ( a diglycoside). On testing for bioactivity, F-11, the bitter saponin (250 ~g ml-') inhibited yeast (Succharoin.yces cereuisiae S11- F, )growth to the extent of 50 - 75% while F, (GOpg ml-') inhibited growth completely. F, and F, were inactive .F, and F-I1 slowed the rate of alcoholic fermentation (F, being mostpotent). But F,: and F, did not have any effect on alcoholic fermentation. However none of the 4 saponins affected the efficiency of alcoholic fermentation (86 - 90%). Antimicrobial studies of flabelliferins using the Bauer - Kirby method showecl that only F, was active inhibiting all bacteria tested, namely; and The stucly is of interest in the area of'structure.activity relationships, as F,, (active) and I?, (inactive) are'isomers'(.hl.W. 868).


INTRODUCTION
Very little work has been done on palmyrah fruit pulp (PFP) although it is available in abundance.' Its free use as a food is detracted by the presence of bitterness. 'A steroid from palmyrah andits monoglucoside andmonorhamnoside were isolated from cultivars from Jaffna. 2 In that study no reference was made 1; o bitterness. A cultivar from Kalpitiya showed the preseilcc of two steroidal glycosides (called flabelliferins) which were labelled F-I and I?-11."-I was a tetraglucoside (M.W. 1080) and F-I1 was a tetraglycoside (M.W.1030) with two rhamilose and two glucose in its carbohydrate moiety."4 The bittei-iless could be removed by the enzyme izaringinase "4 (01-heat stable a-amylase in specimens \ ofPFP from H a m b a n t~t a )~ to give spots at higher Rfvalues. PFPfrorn Hambantota also showed the presence of 3 other flabelliferins called F,, F,, 6z; F,%vhich were isolated by flash chr~matography.~ Flabellifcrins F, & F, were triglycosides (M.W.868) and flabelliferin F, a diglycoside (M.W.722).4 F, was found to be most foam stabilizing and haemolytic . 4 Investigations had also shown that boiled PFP did not ferment well but the ability to ferment was restored by incubating with the debittering enzyme naringinase." Further a s PFP does not spoil easily i t was speculated that the flabelliferins were bioactive. In the present study the action ofthe flabelliferins on growth and alcoholic fermeiltation by a strain of Sacclzaromyces cereuisiae and against G selected bacteria are described. The purpose ofthis study was to determine ifthe isolated flabelliferins have antimicrobial action.

METHODS AND MATERIALS
Palm yrah fruits were collected from Hambantota and pulp extracted manually." The isolation of flabellifel-in was carried out a s described previously."

Yeast growth experiments
Sccccharonz,vces cerevisiae strain S11-F3 was grown on YPD (Yeast Potato Dextrose) slant cultures and inoculated into a synthetic liquid medium" with glucose 3% a s carbol~ydrate source. After 24 h, lini of this culture was centrifuged a t 25°C. The yeast cells were inoculated into 50m1 of the same ~nediurn in a 250ml flask and incubated at 37" C. For growth studics a n initial i~slemocytometer count was obtained. Growth was estimated spcctropl~otometrically a t 660 nm a t 0, 2, 4 and 6 h i n flasks wit11 60 ancl 250 pglinl flabelliferins which was added into the liquid culture prior to sterilization. Controls were used with the same amount of yeast but no flabcllii'erii~s.

Effect of flabelliferins oil fermentation
Fol-alcollolic fermentation the seed culture (25ml) was ccntrifbgcd a t 25OC in a bel~cll centrifuge. After one day (log phase), the yeast was inoculated into a same medium as above but containing 20% glucose and fermented a t 37°C in a 250n11 Erlenmeyer flask for three (3) days, with flabellifcrins (1mglmi)introcluced as above. Controls were also used. The time course for fermei~tativn was followed by measuring CO, evolved estimated by loss ofweight ofthe fermentation f l a s l~.~ The fermentation flasks were fitted and weighed with fermentation bungs and U-tube containing conc. H,SO, (which allowed only evolution of CO, from the system).
Alcohol was determined by the specific gravity method after distillation%ild F residual sugar was determined by the Nelson method."
They were cultured on nutrient agar slants and transferred to growth media containing nutrient agar broth for about 18 h and then spectrophotometrically checked (660 nm) to confirm extent of gr0wth.A.n aliquot (0.1 ml) was spread on nutrient agar plates. Nutrientagar plates had the following composition: nutrient agar, 28 gl-l; agar, 2%. This was used to monitor the effect of flabelliferins ( 62.5 pg -2500 pg) per filter paper disc (9 mm) using the Bauer -Kirby method."' Each flabelliferin was dissolved in alcohol and dried on the 9mm disc. Alcohol (dried with hot air) and ampicillin standard discs (33 pg) were used as controls and as standards. After 24 11, the inhibition zone diameters (in mm) were measured from replicates for each concentration.

Growth studies on yeast
Results are shown in Fig 1,2,3, and 4. Where F-I1 was found to inhibit growth 50-75 % a t 250 pg ml-I and F, completely a t 60 pg ml-l. F, &-F, did not inhibit growth at 250 pg ml-l. All experiments were done in duplicate.

Effect on alcoholic fermentation
Results ( Figure 5,6, 7 and 8) showed that once again Fi, was most bioactive in producing a lag period in fermentation. It was observed ( Table 1) that in all cases fermentation efficiency (conversion of glucose to alcohol taking into account residual sugar) was relatively unaffected ( 86-90% ). A concentration of lmglml flabelliferins was used for each flask. All experiments were done in duplicate.

Effect on bacterial growth
The crude extract F-I1 showed inhibition zones for only three bacteria while F, and F, did not inhibit the bacterial growth (Table 2). However F,, inhibited all the bacterial strains tested (Table 3).

DISCUSSION
Results indicated that flabelliferin F, a steroidal triglycoside was an inhibitor of yeast growth, alcoholic fermentation and bacterial growth. This explains why palmyrah fruit pulp ferments very slowly and does not spoil easily. F,, affected alcol~olic fermelltation by only extending the lag period. The results are interesting as F,, was detected only in samples from Harnbantota. Anotl~cr i~lteresting feature was that both F, (active) and F, (inactive) are isomers (Nl.JV. 868XR It is anticipated that elucidation of the detailed structured features of t l~c carbohydrate moiety of F, and F, will provide insight into structure-bioactivity relationships. Other interesting follow up work include: (1) determination of the diversity of flabelliferins of different known morphological types of tree.
(1 1) determination if any of the products of debittering3 corresponcl to F,.