Screening for Saponins Using the Blood Hemolysis Test An Undergraduate Laboratory Experiment Subramanlam Sotheeswaran School of Pure and Applied Sciences, University of the South Paclfic. Suva, Fiji help of biologists could be sought for the identification of the Saponins are steroidltriterpenoid glycosides. Many sapomaterial collected for examination. nins are found in plants as well as in some marine organisms In one laboratory course a t the University of the South such as sea cucumbers and starfish. Saponins are poisonous Pacific, 21 biological materials were examined. Each student to fish and lower animals. They are produced by marine was assiened one material. The bioloeical materials included organisms for their defense against predators. Plant sapomedicinal plants and marine organisms. The material (10 gJ nins have been shown to have interesting biological activities was extracted with aaueous methanol (7Svc) usinr a Soxhlet such as spermicidal activity (I) and molluscicidal activity extractor. The concentrated extract w& used in the hemoly(2).Saponins from somesea cucumbersshow (3)a high order of activity against pathogenic fungi such as Trichophyton sis test as described below. .. mentagrophytes, Microsporum gypseum, Candida albiTest (5) cans. and Candida utilis. The interesting ~ h a r m a ~ ~ l ~ g i ~Hemolysls al Seven blood-coated agar plates were used in the screening associated with the Chinese drug ginseng, wkch test. Three students were given one plate. Each agar plate is considered a Danacea and a drug for longevity, are attrihcontained five samples: three students' samples, one positive uted to the various saponins present (4) i n ginseng. Plant control (a saponin), and one solvent control (75% MeOHsaponins such as dioscin are commercially sought after as starting materials for the synthesis of steroid hormones. H20). Saponins are therefore, chemically and pharmacologically, Preparation of Blood-Coated Agar Plates an interesting group of natural products. Commercially available blood-agar base (8 g) and distilled Saponins, when shaken with water, reduce the surface water 1200lmLI were sterilized. cooled to 45 O C . Five Dercent tension of water and produce a honeycomb froth. Saponins fresh defihrinated hlood was'added and was mixed thorbehave like soap, thus, their name is derived from the Latin oughly. The blood-coated agar medium was poured into word for soap. All saponins hemolyze blood, that is, they sterilized Petri dishes. The plates can he stored in the refrigbreak down the red blood cells. This property is used in the erator prior to use. screening test for saponins (5).Screening of plant materials or marine organisms has been hitherto confined to pbytoAppllcatlon of Samples chemical and marine research groups. Screening tests for Using a small test tube, cups of hlood agar were removed saponins can he incorporated into undergraduate Laboratory courses. This would enable a large number of biological specfrom the agar plate from five areas of the plate, equidistant from one another (see diagram). Using a 1-mm-diameter imens to be examined for saponins in a short time andalso glass rod, which had been heated previously in a Bunsen would enable undergraduates to learn about such screening techniques. Besides, positive results would mean the discovhurner flame, the agar a t the bottom of each cup was sealed off so that the liquid samples when introduced into the cups ery of new sources of saponins. would not spread beneath the surface of the hlood agar. Collection of Material and ldentlflcatlon Saturated solutions of the extracts from three biological The collection and identification of materials has t o be done by the instructor. About 10 g of each of the biological materials would he necessary for the hemolysis test. The Yleld of Extracts and Hemolysls Zones
-
MBterial
Yield ..of . Methanol Edract*
Hemoly~is Zoneb
Sea cucumber IfoIothwia &I18 (body wail) Bohedschia srgus (body wall) Psolus hbricli (body wall)
0.6
1.3
0.7
1.4
1.5
3.2
Dioscorea esculent&
1.0
1.3
(yam) Derris trifoliahrs
0.9
1.3
Plant material
Diagram: Blood-agar plate with five cups for introduction of samples. 1. CUP lo Introduce solvent Control. 2. CUP lo lntrod~cesaponin control. 3-5. Cups to inhoduce extracts from three materials.
S O a weight ~ psrcent wim respsct to the material. Yiehi 18 B X P ~ ~ 8s bHemolysiomneis indicated by thediotancefrmmeedgsofcuptomelarlhsotendof
the zone (in mm), The hemlyrio mnes for me controls vers 75% MeOH-H20 (nil) and ~poninmmml(0.9 mm). H. eduIIs llocal name: dslm) and D eswlenfa(local name: sarama) are b a h -1 m e n by me lndlgenous people of the South Psclfic Idands.
Volume 65 Number 2
February 1988
161
materials were introduced into three of the cups using separate droppers. The other two cups, in each of the blood-agar plates, were filled with 75% MeOH-Hz0 and a solution of a known saponin, respectively. The Petri dishes containing the hlood agar were covered and were left for a 24-h period, and then the agar plates were observed for any clear zones of hemolysis surrounding the cups. If a hemolytic zone was present, then the distance (in mm) from the farthest point of hemolysis to the edge of the cup was measured. Out of the 21 materials examined by the students, five gave positive results for the hemolysis test. The materials that gave the positive tests and their hemolysis results are given in the tahle.
162
Journal of Chemical Education
Acknowledgment
I am grateful to W. Kenchington for identification of sea cucumbers and to S. Vodonaivalu for identification of medicinal plant materials.
Ll'e'a'ure Cited 1. Kambqj, V . P.Canlroception 1976.14,571. 2. Marston, A,; Hoatettmenn, K. Phytochomistry 1985.24.639. 1 ~ 8 019,1903. , 3. cbrndsl, R.8.;~ ~ ~ t oR.gP.i ,
4. Shiba~,S.~loc.~o~to~kosin~ieentenniol~ointSeminarontheChomisf~lalNoturolPloduccs, Bangkok, Thailand. 1982, p 131.
,,,
,
. , ; Tin-Wa, M.;Farn.worth, N.R. ~ used at university ~ ~ n U.S.A. ~ i ~ ~ i ~ ,
h
~
serening ~ ~M
*~
~ ~ ~ ~~ ~ i I .
