Pretreatment of Urginea sanguinea Bulbs Used in Ethnoveterinary Medicine Influences Chemical Composition and Biological Activity

Abstract

We investigated the effect of freezing on the chemical composition and biological activity of bulbs of Urginea sanguinea Schinz, a known ethnoveterinary medicinal plant. Freshly harvested bulbs were extracted as either fresh material or after oven drying. Frozen bulbs were extracted after overnight thawing at room temperature or after thawing and oven drying. The leaves were extracted dried at room temperature. Plant material was finally ground and extracted with acetone. The yield from frozen material exceeded that from nonfrozen material by more than 10%. The chemical composition of frozen bulbs, as analyzed by thin-layer chromatography (TLC), differed significantly between fresh and dried material and marginally between fresh and thawed material. Because U. sanguinea is used for diseases that could be infection-related, we examined the antibacterial activity of the extracts. The minimal inhibitory concentration against Staphylococcus aureus was 1.25 mg/ml for fresh dried and thawed dried. Other fractions had no substantial antibacterial activity. No extract had significant free-radical scavenging (antioxidant) activity. The mechanism of action in ethnoveterinary use is probably not via antibacterial or antioxidant activity or immune stimulation. Although the dried bulbs were more chemically complex, the extractable mass had decreased from fresh to dried material by at least 13%, possibly because of desiccation. Bulbous material is difficult to extract, and freezing before extraction may be a viable option for use in the herbal industry.

Keywords:

• Antibacterial
• extraction
• freezing
• Staphylococcus aureus
• Urginea sanguinea

Introduction

Therapies of plant origin have been the backbone of human and veterinary medicine for a number of millennia (Cowan, 1999). There have been recent efforts to document ethnoveterinary medicines used in rural small-scale farming in South Africa. (Hutchings et al., 1996; Masika et al., 1997; van der Merwe, 2000; Dold et al., 2001). One such plant used in cattle in the Madikwe area of the North West Province is Urginea sanguinea Schinz, (Hyacinthaceae) locally known as “Transvaal slangkop” (Afrikaans) or “sekaname” (Sotho) (van der Merwe, 2000). The bulb is used either alone or in combination with other plants in the management of a variety of animal diseases, such as blood-borne parasites and gastrointestinal conditions.

Urginea sanguinea is a commonly found invader, distributed throughout South Africa. The plant has a deep red, pear-shaped, onion-like bulb. The entire plant is toxic to animals, yet it is used medicinally (Kellerman et al., 1988). The toxic principle was identified as the bufadienolide transvaalin (Louw, 1952). Animals affected with poisoning show signs typical of cardiac glycoside toxicity. The use of U. sanguinea for treating gastrointestinal conditions may be ascribed to antibacterial activity or may be due to the direct effect of the toxin on the body, which may appear to alleviate the clinical signs associated with the disease.

The objective of the current study was to investigate the antibacterial activity of U. sanguinea. During the initial extraction process, it is difficult to decide which solvent will extract the active components or compounds. This depends on the nature of the compound and the polarity of the extractant. This may be done by using a variety of extractants or by making use of a single “broad spectrum” extraction solvent. Acetone is a good “broad spectrum” extraction solvent, as it extracted the most diverse compounds during initial crude extractions, is easy to use, mixes with water, and was least toxic in the bioassays (Eloff, 1988a).

A large proportion of work done to date on medicinal plants was on dried leaves, bark, and roots. Extraction of fleshy bulbs is more complicated. Urginea sanguinea bulbs can be frozen and planted at a later stage. Because we had frozen bulbs available, we decided to compare the effect of freezing of bulbs against fresh bulbous material on extractability either with or without drying, chemical composition, antibacterial activity, and antioxidant scavenging activity of U. sanguinea.

Materials and Methods

Plant material

Urginea sanguinea was collected from the Onderstepoort Veterinary Institute (OVI). Fresh bulbs with leaves were obtained from the OVI toxicological gardens, and frozen bulbs were obtained from the OVI freezer stores. Prof. C.G. Botha, a toxicologist of the University of Pretoria, identified the bulbs.

Preparation of plants and extraction

The bulbs were analyzed in four different treatments. The freshly harvested bulbs were extracted as either fresh material (F) or after oven drying (FD). The frozen bulbs (kept at − 3°C) were extracted after overnight thawing at room temperature (T) or after thawing and oven drying (TD). The leaves were extracted dried (L) at room temperature.

Frozen bulbs to be extracted dried were thawed and scales separated and dried at 37°C for several days to constant weight (same weight as measured 2 days apart) and were ground to a fine powder in a blender. The powder was extracted with acetone (Merck, technical grade) on a shaker platform at a 5:1 (v:m) ratio. Fresh material was extracted by homogenizing in acetone. The process was repeated three-times after separating the extract by filtration. Each extract was dried, weighed, and made up into a stock solution of 20 mg/ml with acetone.

Analysis of extracts

Five microliters of extract (equivalent to 100 µg dry mass) was loaded on Merck TLC F254 plates and eluted with one of four systems: hexane:ethyl acetate (HE) (2:1); ethyl acetate:methanol:water (EMW) (10:1.3:1); chloroform:ethyl acetate:formic acid (CEF) (5:4:1); or ethyl acetate:hexane (EH) (2:1). These systems separate components over a wide range of polarities. Separated components were marked under visible and ultraviolet light (254 and 360 nm, Camac Universal UV lamp TL-600). All plates were subsequently sprayed with 0.35% vanillin (sigma) (in 5% H₂SO₄ in methanol) with one additional plate, developed in EMW, being sprayed with 5% anisaldehyde (in 5% H₂SO₄ in ethanol). Both were developed at 105°C to optimal color development (Stahl, 1969).

Bioassay

The bioautographic procedure described by Begue and Kline (1972) was used. Thin-layer chromatography (TLC) plates were developed in EMW and CEF, were dried overnight, and were then sprayed with an actively growing strain of Staphylococcus aureus (ATCC 29213) prior to overnight incubation at 100% humidity. Chromatograms were thereafter sprayed with a 2 mg/ml solution of p-iodonitrotetrazolium violet (INT; Sigma). Any clear zones seen after a further 1 h of incubation indicated inhibition of growth. The minimum inhibitory concentration (MIC) was determined using a serial microplate dilution assay (Eloff, 1998b). Staphylococcus aureus cultures were incubated overnight at 37°C with extracts. INT (0.2 mg/ml) was thereafter added to each well. Inhibition was seen by a failure to reduce INT to red tetrazolium. Neomycin (500 µg/ml) was used as the positive control.

Free-radical scavenging activity

The DPPH assay used by Braca et al. (2002) was used. This is an indicator of free-radical scavenging activity. DPPH (2,2-diphenyl-picrylhydrazyl) radical is reduced from a stable free radical, which is purple in color, to diphenylpicryl hydrazine that is yellow. DPPH (0.2% in MeOH) was sprayed onto chromatograms developed in EMW and examined for a color change after 30 min.

Results and Discussion

Quantity extracted

The largest yield extracted was from frozen bulbs (Figure 1; Table 1). It is assumed that the cryogenically induced cellular damage allowed more intracellular components to be extracted. Extraction of cellular polyamines and ions from plants could be increased by first freezing the samples prior to extraction (Minocha et al., 1994). This was ascribed to low-temperature-induced damage to the cell membranes. This effect occurs when freezing was carried out at low freezing temperatures over a period of time, as in standard commercial freezer units (Wolfe & Bryant, 2001).

Figure 1 Percentage yield from different samples.

Table 1 Extraction rate (calculated as % of total mass extracted after same material was extracted three-times) and yield of dry mass based on dry mass of bulbs.

	% Extraction with acetone
Sample	Ex 1	Ex 2	Ex 3	Total yield (%)
Fresh (F)	75.58	18.60	5.81	0.69
Thawed (T)	85.19	11.04	3.76	0.82
Fresh dried (FD)	56.03	30.65	13.32	0.40
Thawed dried (TD)	82.97	13.86	3.17	0.69
Leaves (UL)	65.63	18.75	15.62	2.03

In investigating the extraction rate, extractability was highest in the first extraction. The mass extracted with the first extraction was higher for both thawed samples than for the fresh material, yielding 80% of all material extracted (Table 1). Thus, freezing not only increases the yield extracted but also appears to enhance the efficacy of a single extraction.

Drying substantially decreased the rate of extraction with fresh and thawed bulbs. With fresh bulbs, it decreased the yield by 42%, and with the frozen bulbs it decreased the yield by 16%. It appears that the procedure of drying bulbs before extracting, as is usually done, substantially decreases the yield of compound extracted by acetone.

The dried leaves were included in the study to enable a comparison between yields achieved. The first extraction with the leaves removed approximately 60%. The total yield (%) extracted was, however, double that of the other samples. Thus, it appears that bulbs contain less acetone-extractable material than leaves. The quantity extractable from the leaves of U. sanguinea is up to six-times lower than the quantity extracted from members of the Combretaceae with thinner leaves (Eloff, 1999).

Complexity of chromatographic chemical profile

Because more bands were visible after using the vanillin spray reagent than the p-anisaldehyde spray reagent, only the vanillin spray reagent was subsequently used. There was more than two-times the number of bands from dried material than from fresh material both with fresh and thawed bulbs (Table 2). Extracts from fresh bulbs contained compounds with the same R_f values as extracts from dried bulbs. There was little difference in the number of bands seen under UV light.

Table 2 Total number of spots seen with the different eluents.

	Bands seen
	Vanillin
Extract	EMW	CEF	HE	EH	Total spots
F	4	3	2	1	10
T	4	3	2	1	10
FD	6	5	5	6	22
TD	9	5	5	6	25

CEF, chloroform:ethyl acetate:formic acid (5:4:1); EMW, ethyl acetate:methanol:water (10:1.3:1); HE, hexane:ethyl acetate (2:1); EH: ethyl acetate:hexane (2:1).

TLC is not accurate as a qualitative tool. It may be possible that “inert” compounds such as mucilages may be extractable from fresh material. These compounds may be insoluble upon drying and explains the lower yield from dried bulbs. We decided to investigate the antibacterial and antioxidant activity to determine if the biological activity is affected by the pretreatment of the bulbs.

Biological activity of extracts

Chromatograms developed with CEF and EMW were sprayed with S. aureus to determine which constituent was active. The same single band, which was fluorescent at 254 nm (R_f value 0.89), showed activity for both fresh dried and thawed dried samples. The other two samples showed no activity. The MIC of extracts confirmed the bioautography results (Table 3).

Table 3 The MIC for the various samples cultured with S. aureus.

Sample	MIC
Fresh	>5 mg/ml
Thawed	>5 mg/ml
Fresh dried	1.25 mg/ml
Thawed then dried	1.25 mg/ml
Dried leaves	>5 mg/ml
Acetone	No inhibition
Neomycin	<1 µg/ml

No extract had activity in the DPPH analysis, indicating that U. sanguinea contained no or only low concentrations of antioxidant compounds. In general, plants with phenol compounds possess high antioxidant activity. No phenolics have been reported to occur in U. sanguinea.

Conclusions

The freezing process allowed a greater mass to be extracted, but there was little difference in the number of bands seen when comparing thawed and fresh bulbs. However, more than two times the number of bands were seen when comparing dried bulbs with fresh bulbs. Only the dried material inhibited S. aureus at a MIC of 1.25 mg/ml. Freezing did not affect the in vitro antibacterial activity of the bulbs, and from the bioautography data, the R_f values of the antibacterial compounds were similar.

Differences between dried and fresh material could be due to the high water content within the fresh bulbs (ca. 50%). With extraction at a 5:1 (m/v) ratio, one is more likely extracting with 80% aqueous acetone instead of 100% acetone. It is, however, unlikely that this would have had a major effect on the results.

The fresh material was also slimy, and a large proportion of the extracted material would be mucilage. If mucilage is not antibacterial or does not react with the vanillin spray reagent, the lower number of compounds after TLC may be due to lower quantity on the chromatograph. Similarly the higher MIC activity may be due to the dilution of the “active” compounds by inactive mucilage.

It is not clear whether the effects of drying an artifact in the drying process or whether mucilage present in fresh material had an effect on the extractability of compounds. Intraspecies variation cannot be ruled out (numerous bulbs were used). This was, however, considered non-significant, as all material had originated from the same field, although at different times. Because all plants were exposed to similar environmental conditions, natural phenotypic variation is mostly likely small.

Based on this work, it appears that freezing significantly increases extractable mass. This would be of interest in the commercial herbal industry where maximum yield is important.

Acknowledgments

The section of toxicology at the OVI supplied the plant bulbs. The National Research Foundation funded this research.