Phytochemical Analysis and Toxicity Evaluation of Acetone, Aqueous and Methanolic Leaf Extracts of Agapanthus praecox Willd- Juniper Publishers
Juniper Publishers- Journal of Complementary Medicine
Abstract
Agapanthus praecox Willd of the family
Amaryllidaceae is widely used in South African traditional medicine to
treat heart disease, paralysis, coughs, colds, chest pains and diarrhea.
The decoction is used to prevent pregnancy complications and argument
labor. The study aims at determining the phytochemical constituents and
investigating the potential toxicity of the solvent extracts of A.
praecox. Quantitative phytochemical analysis was done using standard
procedures and spectrophotometric techniques while the toxicity
assessment was carried out with brine shrimp toxicity bioassay. The data
were subjected to two-way analysis of variance (ANOVA) followed by
Tukey’s test. Methanol had the highest percentage yield of extract (16.2
%) while water (9.3 %) was the least. The quantities of tannins
(335.23mg/g-1), phenols (91.11mg g-1), proanthocyanidins (139.47mg/g-1),
flavonoids (46.53mg/g-1) and flavanols (74.02mg/g-1) in acetone extract
were significantly higher (p>0.05) than in aqueous and methanolic
extracts. Highest hatching success was recorded in aqueous extract
(50.33%) and the lowest hatching success (p<0.05) was recorded in the
positive control (amoxicillin). The LC50 of all the solvent extracts
was greater than 1mg/mL (LC50>1mg/mL) while the positive control
(amoxicillin) had a lesser LC50 (0.76mg/mL). Organic solvents of fresh
leaves of A. praecox contained significant phytochemicals that were not
toxic according to Meyer’s index of toxicity, which states that any
substance with LC50>1mg/mL were considered non-toxic. Further
toxicological evaluation assays are recommended to confirm the toxicity
of A. praecox.
Keywords: Agapanthus praecox; Plant extracts; Phytochemicals; Toxicity; Brine shrimp; Hatchability; LethalityIntroduction
The ever-increasing interest in the use of medicinal
plants to treat different ailments and diseases necessitates the
investigation of their potential toxicities. Most people in developing
countries rely on medicinal plants for their primary health care.
Diversity, flexibility, easy accessibility, relative low cost, low
levels of technological input and relative low side effects are some of
the positive features of traditional medicine [1]. All medicinal plants
contain active principles such as saponins, tannins, alkaloids, phenols,
flavonoids, anthocyanins, glycosides etc. which are responsible for
their biological activities [2]. These active principles have been
reported to possess therapeutic as well as toxicological properties [3].
The toxicity of the medicinal plants has been attributed to the
quantities and strength of their active principles [4]. As the global
interest in the use of medicinal plants is increasing, public health
issues and concerns surrounding their safety are also increasingly
recognized. The ethno pharmacological significance of Agapanthus praecox in South African folkloric medicine made it suitable for the current study.
Agapanthus praecox locally called “Isicakathi”
(Isixhosa) is a rhizomatous herb of the family Amaryllidaceae. This
species occurs in Eastern Cape Province of South Africa. It usually
grows
to between 0.8 and 1 metre tall and has 10-11 leathery leaves. The blue
to purple or white flowers are clustered in a large globular flower
head. The fruit is a greenish capsule. It possesses anti-inflammatory,
anti-oedema, antitussive, immunoregulatory, antibacterial, antifungal,
and antitumour properties [5]. It is used in folkloric medicine to treat
chest pains, coughs and colds [6]. Duncan et al. reported its
inhibition of angiotensin converting enzyme in vitro hence, used to
treat high blood pressure and related disorders [7]. The leaves with
Dianthus sp. are used to treat gastrointestinal tract trouble and
abdominal pains [8]. The leaves are used to hold dressings in place and
wound around wrists to bring down fevers [9]. Koduru et al. also
reported the anticancer potential of the root infusion [10].
Agapanthus praecox has been considered a plant
of fertility and pregnancy; it has aphrodisiac property and is used as a
medicine for impotency and barrenness [5]. It has been reported that
the decoction can induce labour and ensure smooth expulsion of the
placenta [11-12]. Pooley stated that “bathing the new born baby with the
decoction of A. praecox makes them strong, keep them free from bowel
problems and prevents crusts on the head” [13]. The anti- depressant
property was reported by Nielsen et al. [14]. It was listed among the
plant species traditionally used
in Southern Africa for the treatment of various central nervous
system-related ailments [15]. Despite, the wide usage of A. praecox
in indigenous medicine system, its phytochemical constituents and
potential toxicity remain unknown. Though, Notten [6] suspected
its haemolytic poisoning in humans but this has not been proven.
To furnish the general public including the traditional medicine
practitioners with adequate information to facilitate better
understanding of the risks associated with the use of this plant;
this study aims at estimating the phytochemical constituents as
well as evaluating the potential toxicity of Agapanthus praecox
using brine shrimp toxicity testing as a preliminary test to
ascertain its level of toxicity.
Materials and Methods
Plant collection
Fresh leaves of Agapanthus praecox were collected from the
University of Fort Hare, Alice Campus, South Africa in June 2016.
The plant was identified by Dr. OJ Sharaibi and a voucher specimen
(SHAMed 2016/07) was deposited in the Giffen’s herbarium of the
University.
Sample preparation and extraction
The fresh leaves were rinsed, air dried and pulverized before
extraction. One hundred gram each of the powdered materials
was soaked in 1000mL of distilled water, acetone and methanol
respectively. The aqueous extract was filtered through Whatman
no.1 paper and the filtrate was freeze-dried for 48 h using a freeze
dryer (Vir Tis benchtop K, Vir Tis Co., Gardiner, NY). The acetone
and methanolic extracts were concentrated to dryness under
reduced pressure at 40℃ using a rotary evaporator (Strike 202
Steroglass, Italy). The resulting extracts were reconstituted in
their respective solvents to give the desired concentrations used
in the study.
Phytochemical Analysis
Determination of total phenol contents
A volume of 0.5mL of the extract (1mg/mL) was mixed with
5mL Folin-Ciocalteu reagent (previously diluted with water 1:9 v/v)
and 4mL (75g/L) of sodium carbonate. The tubes were vortexed
for 15 s and allowed to stand for 30 min at 40 ℃ Absorbance was
measured at 765nm using AJI-C03 UV-VIS spectrophotometer. The
results were expressed as mg/g tannic acid equivalent using the
equation based on the calibration curve:
Y=0.1216x,R=0.9365
Where x is the absorbance and Y was the tannic acid equivalent.
Determination of flavonoids contents
A volume of 0.5mL of 2% AlCl3 ethanol solution was added
to 0.5mL of the sample solution. After 1 h at room temperature,
the absorbance was measured at 420nm. Extract samples were
evaluated at a final concentration of 0.1mg/mL. The results were
calculated as quercetin equivalent (mg/g) using the equation
based on the calibration curve: Y = 0.0255x , R2 = 0.981
Where x was the absorbance and Y was the quercetin
equivalent
Determination of total flavanols contents
The reacting mixture of 2mL each of the extracts with 2mL
of AlCl3 in ethanol solution and 3 mL of 50g/L sodium acetate
solution was allowed to stay for 2.5 h at 20 oC in a water bath. The
absorbance was measured at 440nm. The flavanols contents were
calculated thus: Y = 0.0255x , R2 = 0.9812
where x was the absorbance and Y is the quercetin equivalent
in mg/g.
Determination of proanthocyanidins contents
A volume of 0.5mL each of the plant extracts was added to
3mL of vanillin- methanol (4 % v/v). 1.5mL of hydrochloric acid
was added to the reacting mixture and vortexed. The mixture was
allowed to stand for 15 min at room temperature. Absorbance was
measured at 500nm. Proanthocyanidins content was expressed
as:
Y = 0.5825x , R2 = 0.9277
where x was the absorbance and Y was the cathecin equivalent
in mg/g.
Determination of tannin contents
The extracts weighing 0.20g was added to 20mL of 50 %
methanol, vortexed vigorously and later incubated at 80 oC in a
water bath for 1h. The filtrate was mixed with 20mL of distilled
water, 2.5mL of Folin-Dennis reagent and 10 mL of 17 % aqueous
Na2CO3. The mixture was made up to 100mL with distilled water,
mixed and allowed to stand for 20min. The absorbance of the
tannic acid standard solutions and the sample was measured at
706nm. Results were expressed as mg/g of tannic acid equivalent
using the calibration curve:
Y = 0.0763x , R2 = 0.9644
where x was the absorbance and Y was the tannic acid
equivalent
Determination of alkaloids contents
A volume of 200mL of 10 % acetic acid prepared in ethanol
was added to 5g each of the extracts. This was covered and
allowed to stand for 4h. The mixture was filtered and the filtrate
was concentrated to one-fourth of the original volume in a water
bath. Concentrated ammonium hydroxide was added drop-wisely
to the extract, pending the completion of the precipitation. The
solution was allowed to settle and re-filtered after washing with
dilute ammonium hydroxide. The residue obtained was dried,
weighed and the percentage composition was determined using
the formula:
% Alkaloid= Final weight of the sample / Initial weight of the extract x100 .
Determination of saponins content
The extracts weighing 20g was added to 100mL of 20%
aqueous ethanol and kept in a shaker for 30 min. The mixture was heated over the water bath for 4 h at 55 ℃ and then filtered
to collect the residue which was later re-extracted with 200mL
of 2 % aqueous ethanol. The filtrate was concentrated over the
water bath at 90 ℃ to approximately 40mL. The concentrate was
transferred into a 250mL separatory funnel and extracted twice
with 20mL diethyl ether. The ether layer was discarded while the
aqueous layer was retained and to which 60ml n- butanol was
added. The mixture was washed twice with 10mL of 5 % aqueous
sodium chloride. After evaporation, the samples were dried in
the oven at 40 ℃ to a constant weight. The saponins content was
calculated using the formula:
% Saponins= Final weight of residue/ Initial weight of the sample x100.
Brine Shrimp Toxicity Bioassay
Preparation of the assay system
The assay system was prepared according to Ohikhena, et
al. [16]. Five Petri dishes containing 30ml of the plant extracts
dissolved in filtered seawater were prepared in a two-fold
dilution to yield a series of concentrations (1, 0.5, 0.25, 0.125
and 0.0625mg/mL) of the plant extracts. Amoxicillin dissolved
in seawater (30μl/ml) served as a positive control while Petri
dishes containing sea water served as the negative controls. The
experimental setup was allowed to stand in open air for 30min for
the solvents to evaporate.
Brine shrimp hatchability assay
Artemia salina (Brine shrimp) cysts were stocked at a density
of 10 individuals per Petri dish. Each Petri dish contained 30ml
of the incubation medium at varying concentrations (1, 0.5, 0.25,
0.125 and 0.0625 mg/mL). They were partly covered, incubated
at 28 ℃ and allowed to stand for 72h under constant illumination.
The number of free nauplii in each Petri dish was counted after
every 12h. The percentage of hatchability was assessed by
comparing the number of hatched nauplii with the total number of
cysts stocked. The minimum inhibitory concentration (MIC) was
determined as the minimum concentration of the plant extracts
(or control drug) that inhibited hatching of the cysts.
Brine shrimp lethality assay
The lethality of A. praecox extracts against brine shrimp
nauplii was evaluated by pipetting an aliquot (0.1 ml) containing
10 nauplii into each Petri dish of extract solutions and controls. The
setup was allowed to remain for 72 h under constant illumination;
dead larvae in each Petri dish were counted after every 12 h.
The percentage mortality (M %) was calculated as:
Mortality(%)= Total nauplii- Alive nauplii × 100/Total nauplii
Statistical analysis
Statistical analysis was performed using GraphPad Prism 5
statistical package (GraphPad Software, San Diego MA, USA). The
data were subjected to one-way analysis of variance (ANOVA)
followed by Tukey test. All the results were expressed as mean ±
SEM (n = 3) and were considered statistically significant when p
< 0.05.
Results and Discussion
Dry extract yield
Dry extract yield
Phytochemical constituents
The quantities of tannins (335.2mg/g-1), phenols (91.1mg/g-
1), proanthocyanidins (139.5mg/g-1), flavonoids (46.5mg/g-1)
and flavanols (74.0. mg/g-1) in acetone extract were significantly
higher (p>0.05) than in aqueous and methanolic extracts. The
amount of saponins in all the extracts was considerably while all
the extracts contained little amounts of alkaloids. .
The extracts of A. praecox contained a considerable amount
of phytochemicals that have significant therapeutic and
exhibit biological activities in humans. The presence of these
phytochemicals may be responsible for the diverse uses of this
plant in traditional medicine. The amount of tannins, phenols
and proanthocyanidins in all the extracts were considerably
high. According to Ben Mohamed et al. [19], polyphenols levels
are considerably affected by genetic factors like plant species,
plant organ, phenological stage, and environmental factors
like climatic conditions, biotic and abiotic stresses occurring
during plant growth. Basma, et al. [20] and Chew, et al. [21]
also identified higher amounts of polyphenols in leaves than the
remaining plant organs. The phenolics have been reported to have
antimicrobial, anti-inflammatory and antioxidant activities [22].
Proanthocyanidins have a putative role as antioxidants, they affect
the inflammatory process and protect against H2O2-induced lipid
peroxidation [23]. Yamakoshi, et al. [24] investigated the tox of proanthocyanidins from grape seed extracts and reported
lack of toxicity in both acute and sub-acute toxicity studies and
supported the use of proanthocyanidins as food supplements. The
quantities of saponins in A. praecox leaves in all the extracts were
very high; acetone extract (142%), methanolic extract (132%)
and aqueous extract (123%). Ezeabara, et al. [25] also reported
higher levels of saponins in the leaves of Citrus sinensis and peel of
Citrus aurantifolia. Liu and Henkel [26] considered saponins and
polyphenols as key ingredients in traditional Chinese medicines
which are responsible for most of the observed biological
activities. The saponins in A. praecox leaves may also possess
anti-inflammatory and antibacterial activity which may justify
its usage in the traditional medicine for the treatment of wounds.
The amount of alkaloids in the leaves of A. praecox was very
small (0.296%). The alkaloids present in the leaves of A. praecox
may be responsible for its antimicrobial activity reported in the
literatures; since it plays role in the defence of systems against
pathogens [27].
Brine shrimp hatchability assay
The percentage hatching success of A. salina in different
solvent extracts and the controls were represented in (Figure 1a
& 1b).
Highest hatching success was recorded in the aqueous extract
(50.33%) compared with methanol extract (40.45%) and acetone
extract (40.35%). The positive control, amoxicillin produced the
least hatching success (24%). The order of percentage hatchability
of A. salina cysts was aqueous extract> methanol extract>acetone
extract> amoxicillin. This suggested that aqueous extract had
low inhibitory activity against the A. salina cysts and this may
justify the use of water in herbal preparations. Similar hatching
success recorded in acetone extract (40.35%) and methanolic
extract (40.45%) may be due to the strength and the quantity of the phytochemicals present in these extracts. According to Tulay
[3], the toxicity of the medicinal plants can be attributed to the
strength and quantity of their secondary metabolites.
The hatchability increased with decrease in concentrations
of all the solvent extracts with highest hatching success observed
in 0.0625mg/mL. This may be due to tolerance of A. salina being
at resistant cyst stage to a wide range of salinity and until the
dormancy is broken, hatching will not occur. It may also mean that
all the extracts at 1mg/mL had inhibitory effects and therefore
prevented the cysts from hatching. The increase in the rate of
hatchability with time suggests the nutritive potential of the
extract that the cysts thrived on as incubation progresses. The
highest hatching rate of the cysts for all the extracts was observed
after 72h of incubation. This contradicted Ohikhena et al. [16],
who reported highest hatching success after 36h of incubation
in various solvent extracts of Phragmanthera capitata. The
difference in significant hatching time may be due to difference in
plant species as reported by Tulay [3].
Brine shrimp lethality assay
The results of the percentage lethality of A. salina cysts in
different solvent extracts of Agapanthus praecox, amoxicillin and
sea water were shown in (Figures 2a & 2b).
Among the extracts studied, the highest percentage lethality
was observed in methanol extract (78.53%) compared with
acetone extract (50.22 %) and aqueous extract (42.35 %). Ogugu,
et al. [28] stated that the type of solvent for the extraction process
is vital for the toxicity testing, because different solvents show
different extracting potential. The highest mortality rate observed
in methanol extract can be due to the quantity and the type of
phytochemicals present in the extract; since, methanol had the
highest yield of extract during the extraction process of this study.
The highest mortality rate of A. salina cysts
was observed in
1mg/mL of all the solvent extracts tested. This means increase in
concentration increased the mortality rate and that the LC50 of all the
extracts studied were greater than one (>1), hence the extracts
were not toxic. According to Meyer’s toxicity index, extracts with
LC50 < 1000μg/ml are considered as toxic, while extracts with LC50
>1000μg/ml are considered as non-toxic [29].
The rate of mortality increased with increase in time hence,
the highest mortality rate was observed after 72 h of incubation
of A. salina cysts in all the extracts. The longer the nauplii were
exposed to the plant extracts, the higher the mortality. Significant
mortality observed in all the plant extracts after 72h of incubation
suggests that the plant extracts have some nutrients on which the
nauplii fed on and that the toxic effects of the plant extracts on
the nauplii were delayed but later manifested after a long time of
exposure. This agreed with the reports of Ohikhena et al. [16] and
Otang et al. [30]. According to Ahmed, et al. [31], the toxicity of
plant samples was determined by comparing their LC50 values with
highly toxic substances suitable to be used as positive controls
for this test. In Figure 2b, amoxicillin had the highest percentage
lethality (78.05%) in 1 mg/mL than all the plant extracts and this
showed that amoxicillin used as positive control in this study was
more toxic than all the plant extracts (Table 2).
Conclusion
The different phytochemicals present in the leaf extracts of A.
praecox are of significant biological activities; their presence may
justify the diverse uses of A. praecox in South African traditional
medicine. It was discovered from this study that, all the solvent
extracts of Agapanthus praecox were not toxic (LC50>1mg/mL) in
brine shrimp toxicity bioassay. It was confirmed from this study
that water is the best solvent for preparation of A. praecox as it
poses less risk to the users. Caution should be taken when using
this plant and continuous use should be discouraged. Further
investigation is necessary to determine the toxicity of the methanol
extract of this plant as methanol extract exhibited the highest
toxicity when compared with aqueous and acetone extracts.
Acknowledgment
The authors thank Govan Mbeki Research and Development
Centre, University of Fort Hare, South Africa; for the financial
support given to this study.
Comments
Post a Comment