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juniperpublishers-gjpps · 5 years ago

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Cytotoxic, Antimicrobial and Anti-oxidant Screening of Psidium Guajava Leaves Grown in Oman-Juniper Publishers

Psidium guajava (guava) belongs to the family Myrtaceae, is a most important medicinal plant used in folk medicine to treat gastrointestinal, respiratory disturbances and used as anti-inflammatory medicine. The present study was carried out to evaluate the antioxidant, antimicrobial and cytotoxicity capacities of various crude extracts of the leaves of guava. Dried plant material macerated in ethanol gave crude extract that was Kupchan’s partitioned into hexane, chloroform, and ethyl acetate fractions. The cytotoxicity activity was carried out by brine shrimp lethality bioassay and antimicrobial activity was determined by agar disc diffusion method against E. coli, P. aeruginosa and S. aurues using four different concentrations of each extract including 250, 500, 1000, and2000μg/ml. Cytotoxicity was estimated using brine shrimp test. 2, 2-Diphenyl-1-picrylhydrazyl (DPPH) assay was used for radical scavenging analysis. All extracts did not show any cytotoxicity or antibacterial activity at any of the tested concentration but all extracts showed potent radical scavenging activity, the highest is seen with the hydro-alcoholic and ethyl acetate extracts.Keywords: Gujava Psidium; Crude extracts; Antioxidant; Antimicrobial; Cytotoxicactivity; Oman

Abbreviations: GUAVA: Psidium Guajava; DDPH: 2, 2-Diphenyl-1-Picrylhydrazyl

Introduction

Guava (PsidiumguajavaL L inn.) is commonly known for its food and nutritional values throughout the world. The medicinal properties of guava fruit, leaf and other parts of the plant are also well known in traditional system of medicine since each part of guava tree possesses economic and medicinal value [1,2]. It is distributed in some Arabic countries; Saudi Arabia, Oman and Egypt [3-10]. The biochemical analyses of the crude extracts from leaves and fruits indicated the presence of different group of compounds such as flavonoids, tannins, phenols, triterpenes, saponins, carotenoids, lectins, vitamins, fiber and fatty acids [4,11]. This plant also contains sugar, resins and glycosides [12]. The literature survey reveals that no research has been done on Omani guava. Therefore the present study was to determine the antioxidant, antimicrobial and cytotoxic activity of different leaves crude extracts of guava native to Sultanate of Oman..

Experimental

a. Materials

Hexane, ethanol, chloroform and ethyl acetate were used in this experiment obtained Sigma-Aldrich Company, UK. The other necessary chemicals such as 2-diphenyl-1-picrylhydrazyl, (DPPH), sodium sulfate etc. was obtained from BDH, Germany. Gram positive bacteria (Staphylococcus aureus) and gram negative bacteria (Escherichia coli and Pseudomonas,) were from Biological Department, College of Art and Science, University of Nizwa. Filter paper discs of diameter 6 mm were obtained from Whatmann Company, catalogue number: 8174900. Nutrient ager and plastic Petri dishes were from SharlauChemie Company. Brine shrimp eggs (ARTEMIA CYSTS) were purchased from GOAQUA, Taiwan. Sea salt was obtained from Al-Qurum Muscat. All the glassware used in this present experiment was from Borosil, India.

b. Instruments

UV spectra were recorded on Thermo spectronic spectrophotometer (Great Britain, UK, Model No. Biomate) Ultra speck in methanol (λmax in nm). Rotatory evaporator was used Yamato Rotary Evaporator, Model RE 801, Japan. Incubator used in this experiment obtained from Gen Lab Model: MINO/75F, Serial number: Y5K041.

c. Plant Material

The leaves samples were collected from Alsharqia region, Sultanate of Oman in October 2012. The fresh leave samples were packed instantly after harvesting. The samples were washed with tap water to remove the dust and other foreign particles. The plant was identified and confirmed by the Ministry of Agriculture and Fisheries.

d. Extraction

The samples were dried under shade at room temperature for 7 days. The dried samples (139.45g) were macerated in absolute ethanol (2L) for 7days to give crude extract. The residue was suspended in ethanol/water mixture of 1:1 ratio then extracted successively with hexane, chloroform, and ethyl acetate. All solvents were later removed under vacuum using rotatory evaporator.

e. Anti-bacterial test

The antibacterial test was carried out by agar disc diffusion method [13]. Each extract was subjected to serial dilution technique, using dimethyl sulphoxide as a solvent to give Concentrations of 2000, 1000, 500, and 250 μg/ml. Filter paper discs (6 mm diameter) were Impregnated with each concentration and placed on the agar plates inoculated with the bacteria. Negative controls were prepared using the same solvents employed to dissolve the samples. The plates were incubated micro aerobically at 37ºC for 24 h. Anti-bacterial activity was evaluated by measuring the diameter of the zones of inhibition against the tested bacteria. Each assay was done in triplicate.

f. Brine shrimp test

Brine shrimp (Artemiasalina Leach) larvae were used as indicator animal for preliminary cytotoxicity assay as described by McLaughlin and his group [14]. Shrimp larvae were hatched in artificial sea water prepared by dissolving 38g of sea salt in distilled water (IL). The sea salt was placed in a small tank divided into two compartments byPerforated polythene wall. About 50mg of GOAQUA brine shrimp eggs were sprinkled at covered chamber of duo compartment plastic container. The open compartment was illuminated to attract the shrimp larvae from the dark compartment once were hatched within 24 hours.

g. Brine shrimp lethality test

Solutions corresponding to 10, 100, 250, 500, 750 and 1000 mcg/ml were prepared in six vials by serial dilution of the stock samples (10mg/ml).Each experiment was done in triplicate. A total of 10 larvae were transferred in each vial and the solutions were diluted to 5 ml by adding the artificial sea water. Mean percent mortalities of the larvae were calculated after 24 hours of exposure.

h. Radical scavenging activity using DPPH method

Free radical scavenging activity of different organic extracts was estimated as described by Blois [15] with minor modification. Four concentrations (12.5, 25, 50, 100 and 200 ppm equivalent to 12.5, 25, 50, 100 and 200 μg/ml, respectively) were prepared for each extract (hexane, chloroform, ethyl acetate and hydroethanol. Four ml from each concentration were placed in a test tube to which one milliliter of 0.1 mM methanol solution of DPPH (2,2-diphenyl-1-picrylhydrazyl) was added and shaken vigorously. After that all the test tubes were allowed to stand at 27 ºC in dark place for 45 min. The control was prepared in the same way but without adding extract. The absorbance of the prepared samples was measured using UV spectroscopy at 517nm. Radical scavenging activity of the tested crude extracts samples was estimated as the inhibition percentage and was calculated by using the following formula,

Results and Discussion

a. Anti-microbial studies

Previous studies which were done by Gonclaves, et al. [4] showed that there was some activity against S.aureus on the hexane extract, ethyl acetate extract, and methanol extract, but at high concentration of the three extract showed activity against E. coli and only the methanolic extract showed activity against Salmonella spp [4]. In a study that was done in Jordan, Psudium guava acetone extract showed sensitivity to Provenciastearti, Providenciarettgeri, Strepoccus group c, Staphylococcus auress, Candidealbicans. There was no activity against Proteus vulgaris, E. coli, Salmonella ssp, Pseudomonas aeruginosa, Streptoccusfaecalis found [12].Penecilla, et al. [13] in the Philippines showed that the leaves acetone, ethanol and aqueous extracts had inhibitive activity but not for the hexane extract [13]. The four extracts hexane, chloroform, ethyl acetate and hydro alcoholic didn’t show activity against gram +ve or gram -ve bacteria. This variation from other studies might be attributed to variation in environment, including day length, light intensity, ambient temperature, rainfall, soil or season of collection. All the crude extracts did not show any mortality at any concentration.

b. Anti-oxidant activity

The radical scavenging activity of the different crude extracts of guava leaves are presented in Figure 1. All extracts showed high radical scavenging activity at 50μg/ml concentrations, the hydro-alcoholic and the chloroform extract exhibited more scavenging effects on free radicals than did the hexane and ethyl acetate extract. On the basis of the results obtained, guava Hydro-alcoholic and chloroform extracts the leaf of Guava can be used for a variety of beneficial chemo-preventive effects. However, further studies on the antioxidative components of guava extracts are required.

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juniperpublishersjcmah · 5 years ago

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Phytochemical Analysis and Toxicity Evaluation of Acetone, Aqueous and Methanolic Leaf Extracts of Agapanthus praecox Willd- Juniper Publishers

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; Lethality

Introduction

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

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. .

Values are the mean of the three replicates (n=3).

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.

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juniperpublisherscellscienc-blog · 5 years ago

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Evaluation of antioxidant and cytotoxic properties of Vernonia amygdalina- Juniper Publishers

Abstract

The present investigation was carried out to evaluate the antioxidant activity and cytotoxic properties of Vernonia amygdalina. The free radical scavenging activity using a stable radical; 2, 2-Diphenyl-1-picryl hydrazyl, lipid peroxidation assay (DPPH), and nitric oxide inhibitory assay gave the highest percentage inhibition as 74.55±1.07%; IC50 = 1.831, 60.42±0.11; IC50 = 3.84 ± 1.03 and 71.26±0.48; IC50 = 0.99mg/ml, respectively. This is comparable to the standards quercetin used (P>0.05). In addition; total phenol, total flavoniods, anthocyanin and proanthocyanidine of the extract were determined using established methods. The results obtained justify the scavenging activity of the extracts. Furthermore, the extracts possessed very low cytotoxicity to brine-shrimp lethality test, when compared with the reference standard (Potassium dichromate, LC50 = 0.003±μg/mL). The results obtained in the study indicate that V. amygdalina can be a safe potential source of natural antioxidant agent; used as a neutralcetical/functional food..

Keywords: 2; 2-Diphenyl-1-picryl hydrazyl; Antioxidant; Cytotoxicity; Veronia amygdalinais

Introduction

Vernonia amygdalina is a shrub that grows predominantly in the tropical Africa. Leaves from this plant serve as food vegetable and culinary herb in soup [1]. Anecdotal evidences suggest the use of V. amygdalina in the treatment of feverish condition, cough, constipation, hypertension and related vascular diseases as well as diabetes. Photochemical screening of this plant leaves extracts showed the presence of Saponins, riboflavin, polyphenols, sesquiterpene and flavonoids [2]. Strong antioxidant activities involving flavonoids extracted from V. amygdalina and its saponins have been reported to elicit anti-tumoral activities in leukemia cells [3]. In addition, peptides from V. amygdalina are known to be potent inhibitor of mitogen activated protein kinase (MAPK) which is involved in the regulation and growth of breast tumour [4].

Previous studies have shown that a good number of plants have antioxidant activities that could be therapeutically beneficial. Consequently, antioxidant agents of natural origin have attracted special interest because of the potential they hold in the maintenance of health and protection of some age related degeneration disorders, such as coronary heart disease and cancer, neurodegenerative disease [5-7].

Although, antioxidants from natural sources are beneficial, it is pertinent to know their bio-safety. In this regard, the brine shrimp lethality assay is considered a useful tool for preliminary assessment of toxicity of plant extracts; a suggested pharmaco logical screening method in plant extracts. It has been used for the detection of fungal toxins, plant extract toxicity. The shrimp lethality assay was proposed by Michael and co-workers in 1956, and later developed by Vanhaecker and his group in 1981. This is based on the principle, whereby the kill laboratory-culture of an invertebrate, Artemia salina L (the brine shrimp larva) following exposure to a varied concentration of plant extracts, heavy metals, cyan bacteria toxins and pesticides, is assessed for toxicity [8]. The purpose of this study is to evaluate the acute toxicity and antioxidant properties of V. amygdalina in relation to its use as a neutralcetical.

Materials and Methods

V. Amygdalina: Fresh leaves of V. amygdalina were collected from the University Village, Kogi State University, Nigeria. The plant material was identified and authenticated by taxonomist in the Department of Botany, Kogi State University, where the voucher specimen (VA-111) was deposited. Fresh leaves of V. amygdalina were air dried under room temperature until a constant weight was obtained. Thereafter, the leaves were milled to a coarse powder with the use of laboratory Mortar and Pestle. After this, 20g of the plant powder was weighed into a volumetric flask and then extracted using 200mls of distilled water for 72 hours. The crude extract was obtained by concentrating the water soluble extract using rotary evaporator at 45 °C. The working solution of extract was prepared by weighing out 0.02g of crude extract accurately and dissolved it in 20ml of distilled water to give an effective concentration of 1mg /ml.

Radical scavenging activity

In order to determine the antioxidant properties of the plant, radical scavenging activities of the leaves extract, was determined using the stable radical DPPH (2, 2-diphenyl-1 piccrlhydrazyl hydrate) according to the method of Blois (1958) as describe by Babalola and co-workers [9]. The principle is based on the reaction of DPPH, and an antioxidant compound to generate hydrogen, which is reduced (DPPH + RH → DPPH2 + R). The observed colour change from deep violet to light yellow was measured at 517nm. To 1ml of varied concentrations (0.5, 0.25, 0.125, 0.0625, 0.003125mg/ml) of the extract or standard, was added 1ml of 0.3mM DPPH in methanol. The mixture was vortexed, and then incubated in a dark chamber for 30minutes. Thereafter the absorbance was read at 517nm against a DPPH control containing only 1ml of methanol in place of the extract. The antioxidant activity (AA) was then calculated using the formula:

AA = [(Ao – Ac)/Ao] x 100,

Where: Ao = absorbance without extract and Ac = absorbance with extract.

Nitric oxide

Sodium nitroprusside generates nitric oxide in aqueous solution at Physiological pH, which consequently interacts with oxygen to produce nitric ions. This was measured by Griess reaction [10].

Procedure: 3ml of the reaction mixture containing sodium nitroprusside (10mM) in phosphate buffered saline (PBS) together with the varying concentrations of the extract (0.5, 0.25, 0.125, 0.0625, 0.003125mg/ml) were incubated in a water bath at room temperature for 150 minutes. This was followed by the removal of 1.5 ml of the reaction mixture and the addition of 1.5 ml of Griess reagent. After which, the absorbance of the chromophore formed was read using spectrophotometer at 546nm. Percentage inhibition of nitric oxide radical by the extract was calculated using the formula:

NO = [(1-E/C)] x 100,

Where: C= absorbance value of the fully

Ferric reducing antioxidant power assay (FRAP) assay

The FRAP assay used antioxidants as reductant in a redox linked colorimetric method with absorbance measured with a spectrophotometer. A 300mmol/L acetate buffer of pH 3.6 (3.1g of sodium acetate+16ml of glacial acetic acid made up to 1L with distilled water, 10mmol/L 2, 4, 6-tri (2-pyridyl 1, 3, 5-triazine, 98% (sigma-Aldrich) (3.1mg/ml in 40mmol/L HCl) and 20mmol/L of ferric chloride were mixed together in the ratio of 10:1:1, respectively to give the FRAP working reagent.

Procedure: A 50μL aliquot of extract was added to 1.5ml of FRAP reagent in a semi-micro plastic cuvette. Absorbance measurement was taken at 593nm (A593) exactly 10 minutes after mixing using 50μL of water as the reference. Thereafter, to standardize 50μL of the standard, iron (III) sulphate, (1mM) was added to 1.5ml of FRAP reagent. All measurement was taken at room temperature in the absence of light.

Evaluation of total phenolic content

The total phenolic of V.amygdalina extract was determined using the folin ciocalten assay method of Singleton and Rossi (1965) [11]. To 0.1ml of 1mg/ml of extract /standard was added 0.9ml of distilled water. Thereafter, 0.2ml of folic reagent was added. This was vortex-missed. Subsequently, 1ml of 7 % Na2CO3 solution was added to the mixture after 5minutes. The solution was followed by dilution to 2.5ml and then incubated for 90minutes at room temperature. The absorbance was read at 750nm against the reagent blank. Standard preparation was carried out by preparing a stock solution of gallic acid (1mg/ml) aliquots of 0.2,0.4, 0.6,0.8 and 1ml were taken and made up to a total volume of 2ml.

With the equation as shown below, the total phenolic content of the plants was then calculated, and expressed as mg gallic acid equivalent (GAE)/g fresh weight. The analysis was carried out in triplicates.

Equation (1) - - - - -C=c *v/m

Where: C = total content of phenolic compound in gallic acid equivalent (GAE); c = concentration of gallic acid established from the calibration curve, mg/ml; V=volume of extract (ml); m = Weight of the crude methanolic plant obtained

Evaluation of total flavonoids content

Aluminium chloride colorimetric method described by Zhilen was used for the determination of the total flavonoidal content of the plant extract [5]. Water (0.4ml) was added to 0.1ml of extract/ standard, as well as 0.1ml of 5 % sodium nitrite. This was left for 5minutes. Thereafter, 0.1ml of 10 % aluminium chloride and 0.2 ml of sodium hydroxide was added to the solution, and the volume was adjusted to 2.5ml with water. The absorbance at 510nm was measured against the blank.

Standard preparation

A stock solution of quercetin (1mg/ml) was prepared. Aliquots of 0.2, 0.4, 0.6, 0.8, and 1ml were taken and the volume made up to 2ml with distilled water.

The total flavonoid content of the plant extract was then calculated as shown in the equation below and expressed as mg quercetin equivalents per gram of the plant extract. The analysis was conducted duplicates and mean value considered. X = q×V/w: Where X= total content of flavonoid compound in quercetin equivalent; q = concentration of quercetin established from the standard curve; V=volume of extract (ml); w = weight of the crude methanolic extract obtained.

Proanthocyanidin content determination

The proanthocyanidin content of the extract was determined spectrophotometrically [12]. Extracts were diluted to provide a spectrophometric reading between 0.1 and 0.8 absorbance units.

Procedure: A 0.25ml sample aliquot of adequately diluted extract was added to 2.25ml of concentrated hydrochloric acid in n-butanol (10/90, v/v) in a screw top vial. The resulting solution was mixed for 10 to 15 seconds. Extracts were then heated for 90 minutes in an 85 °C water bath then cooled to 15-25 °C in an ice bath. The absorbance at 550nm was measured on a UV visible spectrophotometer. A control solution of each extract was prepared to account for background absorbance due to pigments in the extracts. The control solution consisted of the diluted extract prepared in the hydrochloric acid/n-butanol solvent without heating.

The proanthocyanidin content was expressed as mg cyaniding per Kg of sample.

Where:

ΔA = A550sample – A550control

A550 sample = Sample absorbance at 550nm

A550control = control sample absorbance at 550nm

Є = Molar absorbance co efficient of cyanidin (17,360L-1M- 1cm-1)

L= pathlenght (1cm)

MW= Molecular weight of cyaniding (287g/mol)

DF= dilution factor to express as g/L

1000 is the conversion from grams to milligram

Determination of total anthocyanin content

Total anthocyanin content of the extract was determined by the pH differential method [13].

Procedure: A pH 1.0 buffer solution was prepared by mixing 125ml of 0.2 N KCl with 385 ml of 0.2 N HCl and 490ml of distilled water. The pH of the buffer was adjusted to pH 1.0 with 0.2 N HCl.A pH 4.5 buffer solution was prepared by mixing 440ml 0f 1.0 M sodium acetate with 200ml, 1.0M HCl and 360ml of distilled water. The pH of the solution was measured and adjusted to pH 4.5 with 1.0 MHCl.

0.5ml of the extract was diluted to12.5ml in the pH 1.0 and 4.5 buffers, and allowed to equilibrate in the dark for 2 hours. The absorbance of the samples at 512nm (A512nm) and 700nm (A700nm) was measured on a UV- visible spectrophotometer. The difference in absorbance (ΔA) between the anthocyanin extract diluted in pH 1.0 and pH 4.5 buffers was calculated using the equation below

ΔA= (A512 pH1.0-A700nm pH1.0)-(A512nm pH4.5-A700nm pH 4.5)

The A700nm was employed in the calculation of ΔA to correct for any background absorbance due to turbidity on the extracts. The anthocyanin content was expressed as mg cyaninidin 3-glucoside per 100g berries using a molar absorbance co efficient (Є) of 26900 L-1M-1cm-1(Guisti and Wrolstad, 2001).

TACY = (ΔA×MW) ×DF×1000

Є ×0.1×1

Where:

TACY= Total anthocyanin expressed as mg cyaniding 3-glucoside/ 100g of plant material

MW= molecular weight of cyaniding 3-glucoside (449.2g/L)

DF= dilution factor to expressed the extracts on per gram of plant basis

Є= molar absorption co efficient of cyaniding 3-glucoside (26900 M-1cm-1)

0.1= is the conversion factor for per 1000 grams to 100 grams basis.

Brine shrimp bioassay

Brine shrimp lethality test was carried out using hatched Brine shrimp (Artemia salina L) larvae (nauplii) according to the procedure described by The eggs were hatched in artificial sea water (16g of sea salt in 50ml of distilled water) by adding 100mg of brine shrimp eggs to 50ml of sea water that was partitioned into two compartments. The compartment sprinkled with the cysts was left dark, while the other compartment was supplied with bright white fluorescent light. After 24hours of incubation, the hatched shrimps moved to the illuminated side. Ten brine shrimps larvae were then counted and transferred to each sample vial, using a Pasteur pipette and artificial sea water was added to make 10ml. The sample vials were previously containing solution of the extract prepared by dissolving 0.2g of the extract in 20ml distilled water to give concentration of 1mg/ml. The varied concentrations from the stock solution were transferred to different graduated container with the aid of a micropipette. The survivors were counted after 24 hours. Three independent studies were carried out (n =3).

Statistical Analysis

The results are expressed as mean±SEM using Graph Pad Prism Graphical-Statistical Package version 5. The difference between groups was analyzed by Student t-test followed by Dennett’s test with 5% level of significance (p<0.05).

Results

Antioxidants

The extract was assayed for total content of four major types of antioxidant properties. The antioxidant constituents were: to tal phenol, total flavonoid, proanthocyanidins and anthocyanins. However, the percentage yield of the crude extract used for the assays is given as 10.11±1.08%. The results showed the total phenolic content as 1.588±0.04mgGAE/g, which is considerably high compared to the standard. The total flavonoid content expressed as quercetin equivalent per gram of the plant extract showed that the test material had 0.857±0.15mg QUE/g dry weight for the crude extract (Table 1). These two indices are pointer to an increased antioxidant activity. The concentration of anthocyanin in the sample was 0.099±0.08 cyanidin 3-glucoside/100g for the crude extract, while the concentrations of proanthocyanin was 0.038±0.05 cyanidin 3-glucoside/100g for the crude extract. Tannin was also assayed, and it gave a concentration of 1.188±0.04mg/ml (Table 1).

All values are expressed as mean±SEM (n=3)

Antiradicals

The result of the antiradical assays carried out on the extract is shown in Table 2. Using the DPPH (2, 2-diphenyl-1-piccrlhydrazyl hydrate) assay, a well established antiradical assay, the activity was concentration dependent i.e. activity increases with increase in concentration. The extract gave the highest inhibition of 74.55±1.07% at 0.005mg/ml. The calculated IC50 values for the test extract and standard Quercetin were 1.831±0.15 and 0.00326±0.24mg/ml, respectively Table 2. The extract used showed activity despite the significant difference (P<0.05) between the test and standard.

All values are expressed as mean±SEM (n=3). The level of activity between the crude extract and the standard Quercetin is significantly different (p<0.05)

All values are expressed as mean±SEM (n=3).

The nitric oxide inhibition assay also showed that V.amygdalina is a potent scavenger of nitric oxide as shown by the percentage inhibition and IC50 of 3.84±1.03mg/ml Table 3. The FRAP assay result showed a concentration dependent change when the FRAP values of the test fractions were determined. Results were expressed in mmol Fe2+/L. The concentration of Fe2+ in the reaction mixture at 0.5mg/ml, was given as 1.49±0.18 mmol Fe2+/l for the test extract (Table 4).

Brine shrimp lethality test

As shown in Table 5, the plant extract showed the highest percentage lethality to be 75% with LC50 of 1.49mg/ml, whereas, the LC50 for the positive standard (K2Cr2O7) was found to be 10.91±2.22μg/ml. The plant extract showed concentration at 50% percentage lethality to be a little greater than 1mg/ml compared to the standard. In essence, the test sample at the concentration used could be harmless to the biological system. All values are expressed as mean±SEM. This result is a triplicate of three independent experiments.

Discussion

Studies have shown that consumption of biosafe exogenous and natural antioxidant is beneficial, as regard combating diseas es such as cancer, arthritis, diabetes, among others. These diseases emanates from oxidative stress mostly caused by reactive oxygen species (ROS) [14-16]. Moreover, synthetic antioxidant, including tert-butylhydroquinone (TBHQ), buthylatedhydroxytoluene (BHT) and propylgallate have been found to be beneficial, but toxic, as well as with attendant effects [17,18]. This is shown by comparing the bio-safe syzygium cumini fruit juice, a natural antioxidant to the toxic BHT on serum enzymes such as ALT (alanine transferase), AST (aspartate transferase), alkaline phosphatase and urea in rats [19]. For this reason, it has become imperative to continue to investigate and search for more bio-safe antioxidants that could be relevant in the fight against oxidative stress V. amygdalina is useful in this regard [20-22]. Kahaliw and his group have reported on the biosafety of this plant [23]. Moreover, anecdotal evidence attests to its use in the treatment of different ailments after boiling, as well as its use in the preparation of soup. This informed the aqueous extraction carried out, as opposed to the use of organic solvents, such as methanol and ethanol.

A lot has been reported on V. amygdalina as a functional food. In order to further establish its biosafety, the result in table 5 and the work of Kaali justifies V. amydalina as an anti-malaria agent that is biosafe for all the benefits discoursed above [29]. The study of Patnaik and Bhatnagar is in agreement with this study [30]. Moreover, Thompson showed comparable results [31] Data from alcoholic extract of V.amygdalina [32,33] is statistically indistinguishable compared to this study (Table 5).

Conclusion

On the basis of the data from this current research, V. amygdalina is a potent antioxidant attributable to their flavanoid and phenolic constituent that is biosafe for all the health benefits that is known for.

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