Antimicrobial and cytotoxic potential of seeds and flowers crude extracts of sunflower

Nuzha Bint Mahdi Bin Ali Al-Shukaili,Mohammad Amzad Hossain

College of Pharmacy and Nursing,University of Nizwa,Nizwa 616,Sultanate of Oman

Keywords:

Helianthus annuus

Sunflower

Antimicrobial potential

Cytotoxic potential

Agar gel diffusion method

BSL method

A B S T R A C T

Oil-producing crops,including Helianthus annuus L.(H.annuus,Sunflower),have been used for the treatment of different human diseases since ancient times.This study aims to determine the antimicrobial and cytotoxic potential of different polarities of seeds and flower extracts of locally grown H.annuus by well-established bioassays.To achieve the objects,the plant extracts were prepared from both flowers and seeds powder samples individually with methanol solvent by using a Soxhlet extraction method.The antimicrobial and cytotoxic potential of the flowers and seed extracts were determined by the in vitro diffusion and brine shrimp method(BSL).The results of the antimicrobial potential of both prepared flower and seed extracts of various polarities at different concentrations showed promising potential against the gram-positive and gram-negative bacterial strains,within the range of inhibition zone 0-20 mm.Among the flower extracts,the highest potential was obtained in water extract and the lowest potential was in butanol extract followed by water ＞ethyl acetate ＞hexane ＞methanol ＞chloroform ＞butanol extracts.However,from the seed extracts, the highest potential was obtained in water extract and the lowest potential was in the methanol extract followed by water ＞hexane ＞butanol ＞ethyl acetate ＞chloroform ＞methanol extract.On the other hand,the results of cytotoxicity among the flowers extracts against the brine shrimp method where the highest LC50 against brine shrimp nauplii were found to be 31.25 μg/mL in water extract and the lowest LC50 was 1345.86 μg/mL in chloroform extract followed by water ＞ethyl acetate ＞hexane ＞butanol ＞methanol ＞chloroform extract.Similarly,different polarities of seed extracts,the highest LC50 against brine shrimp nauplii was shown to be 109.56 μg/mL in butanol extract and the lowest LC50 was 1513.56 μg/mL in ethyl acetate extract followed by butanol ＞hexane ＞water ＞methanol ＞chloroform ＞ethyl acetate extract.In conclusion,the results showed that both flowers and seeds extracts at various concentrations have promising potential against the positive and negative bacterial strains and the highest potential water extract could be used as natural broad-spectrum antibiotics as herbal medicine to treat different human infectious diseases.

1.Introduction

Scientific evidence proposes that eating more fruits and vegetables has a lesser hazard of cancer[1].Polyphenols and their derivatives are important for plant metabolites,which is most important for the human diet.Besides,the polyphenols and derivatives play a wide variety of pharmacological and phytochemicals functions.The biological functions are directly or indirectly associated with interference in several stages of cancer growth[2].

H.annuus L.(H.annuus,Sunflower)is an economic crop that originated in North America.H.annuus belongs to the Asteraceae family[3].The selected species is an annual flowering plant and native to American tribes who domesticated the crop(1000 BCE)and it was transferred towards eastward and southward America.The first cultivation of H.annuus(sunflower)was in southern Canada and Mexico but nowadays,the selected plant is cultivated on a commercial basis all over the world[4].The Arabic name of this species is Eibad Alshams.It is usually grown everywhere in all climates for its edible oil and fruits [4-6].The selected plant species is widely branched with many flowers[6,7].The color is yellow,red,orange with sexually sterile.The selected sunflower plant is considered as an oil-producing crop.The seeds contain oil, and the oil is isolated from the seed by pressed[7-11].Several studies are showing that H.annuus extracts are a rich source of carbohydrates,flavonoids,toxic alkaloids,saponins glycoside, phytosterols,steroids, and oils were present [12-18].In addition,the sesquiterpene lactones,the correct heliangolide niveus in B and its ethoxy derivative were also present in the selected species.Other critical chemical compounds found in the sunflower such as vitamin B, oxalic acid,vitamin C,caffeic acid,chlorogenic acid and dicaffeoylquinic acid which are also responsible for biological potential[12-18].The H.annuus extracts by organic solvents showed potential antimicrobial potential against different Gram(+)and Gram(−)bacterial strains[11].About 53%of the total world H.annuus is produced in Arabian countries,and 0.5%of the total area harvested[6].In Oman,various species of this family are available here,and it is used traditionally as diuretic and expectorant[11].

Traditionally,the leaves tea is used as astringent,diuretic and expectorant,and it is also used to treat high fevers[19,20].The dried powder leaves of the selected plant are used for the treatment of poultice,swellings,snakebites,and spider bites[21].Traditionally,the tea infusion of the flower is used for the treatment of malaria and lung ailments and the head of flower and seeds are used to treat febrifuge,nutritive and stomachic[22].The matured seed is traditionally used as diuretic and expectorant; the literature reported that plant had been used to treat many pulmonary complaints[23].The roots paste is used to treat rheumatic aches and pains[19].The selected species is oil-producing plant and its seeds contain up to 45%oil and the oil is used often with drying oil such as linseed to prepare different household items like making soap, candles, varnishes, paint, as well as for lighting [22,23].Too much work has been done and reported on the different species of sunflowers.However,in Oman,still,there is a lack of information about the selected plant species, which is grown in Oman.Therefore, this study aims to prepare the crude extracts from flowers and seeds and to determine their antimicrobial and cytotoxic potential by using agar gel diffusion and BSL methods.

Researchers are working on plant sources to find naturally occurring safe antibiotics for the use of medicine instead of synthetic antibiotics.The Sultanate of Oman is known for its plant diversity,and plenty of several rare plant resources are available in Oman.The available plants here are rarely still unknown or undetermined.The selected species is used locally to treat various diseases.Urgently,the novel and effective drugs from the plant sources are needed to search that may prevent or treat diseases without any side effects.Therefore,this present study design is to prepare various extracts from the flowers and seeds of H.annuus and to assess their cytotoxic and antimicrobial potential by the usual technique with slight alteration;these various extracts from the flowers and seeds of H.annuus were collected from Barka,Muscat,Oman.

2.Materials and methods

2.1.Microorganism

Five pathogenic microorganisms were used in this experiment such as Gram-positive Staphylococcus, Streptococcus, and S.aureus and Grams-negative E.coli and Klebsiella harmful bacteria were collected from Nizwa Hospital,Nizwa,Oman at the end of December 2018.The collected bacterial strains were cultured in the Biology Laboratory,College of Arts and Sciences,Nizwa University,Nizwa for carrying out our present experiment.

2.2.Sample collection

The samples of seeds and flowers were collected from the local farmers of Barka, Muscat, Oman.The samples were collected twice.The seed samples were collected at the local Supermarket on October 16, 2018, in the morning times.However, the flowers were collected from the farmers at the end of January when the flowers were blooming.The flower samples were collected around 10 am.Both collected seeds and flowers samples of H.annuus were brought home for washing,and drying[24].

2.3.Preparation of crude extracts and fractionation

The flowers and seed samples were rinsed to discard undesirable particles and the samples were dried inside the room by using an electric fan at ambient for five days.Later on,the dried samples were crushed into coarse powder individually by using a massive duty blender machine.The coarse seed powder samples(300 mg)were packed into a filter paper and introduced into a thimble tube.It was extracted with methanol(2 L)by using a Soxhlet apparatus and recycled the process for several days.After the completed time,the samples were filtered,and the methanol solvent was evaporated dryness by a rotary evaporator at 40°C[24].The weight of methanol extracts of flowers and seeds was 36 mg and 60 mg,respectively.Finally,the flower and seed crude extracts(35 and 50 mg)were dissolved in water(200 mL)separately and then it was transferred to a separatory funnel for fractionated by using different polarities organic solvents with increasing polarity to give the corresponding hexane,chloroform,ethyl acetate, butanol, water extract, which was used for the evaluation of antimicrobial and cytotoxic potential.Similarly,the seeds extract was fractionated by using various polarities with increasing polarity pattern to give the corresponding extracts.

2.4.Antimicrobial potential

Both prepared flower and seeds extracts of different polarities at various concentrations were used to evaluate antimicrobial potential by using the disc diffusion method against culture pathogenic gram-positive and gramnegative bacterial strains[24].Levofloxacin and DMSO were used as a positive and negative control.The concentration of positive control was 1 mg/3 mL in DMSO.In this experiment,six-millimeter diameter(6 mm)was used as discs, and the discs were made from Whatman filter paper by using a punch machine.For the determination of antimicrobial potential at four different concentrations such as 500, 250, 125 and 61.5 μg/mL were used of each polarity extract.Initially,all the discs were sterilized and then soaked with each concentration extract and place on the inoculated agar plates.All the sample-loaded plates were incubated at 37°C for 24 h.The diameter zone of inhibition of each loaded plate against the selected bacteria strains was measure.The experiment was replicated three times.The antimicrobial potential index for each extract was calculated by using the following established formula:

Potential index AI( )

=Inhibition zone of the sample/Inhibition zone of the standard.

2.5.Cytotoxic potential

The cytotoxic potential of methanol and its derived various polarities extracts at different concentrations was used for the determination of toxicity through the brine shrimp lethality method[25].Initially,the shrimp eggs were hatched at a closed container containing seawater for 24 h.After 24 h,the shrimp was hatched and the nauplii came out and separated from the shrimp eggs.In this experiment,the hatched nauplii were used to evaluate the cytotoxic potential.Five different concentrations such as 500,250,125,62.5,31.25 μg/mL of methanol,and its fractionated extracts were prepared by using water.Each of these test solutions,a 100 μL sample was placed in test tubes containing 4.9 mL of seawater and ten nauplii.Then,all the tested tubes were incubated for 24 h under the light.After incubated,the number of surviving nauplii in each test tube was counted and noted.The IC50was calculated for each concentration of the sample.

3.Results and discussion

Still,the plant and its formulated products are used as primary sources of healing system worldwide.However,since the old-time,researchers are interested in working on plant sources to find an effective therapeutic agent.The research findings showed that the plant crude extracts are more effective than the synthetic ones with no or insignificant side effects[21-23].However,a few scientific types of research on plant sources and their biological potential have been worked out.On the other hand,the resistance to microbial growth by using multidrug,which is,bared the effectiveness of synthetic drugs [26,27].The H.annuus extracts by organic solvents showed potential antimicrobial potential against different Gram(+)and Gram(−)bacterial strains[28].About 53%of the total world H.annuus is produced in Arabian countries,and 0.5%of the total area harvested[6].In Oman,various species of this family are available here,and it is used traditionally as diuretic and expectorant.However,still,there is no scientific work done on Omani species by the Omani researcher.Therefore,this current study design to prepare various polarities extracts from flowers and seeds and to assess their antimicrobial and cytotoxic potential by using the standard protocol, which was collected from the local farmer and supermarket.

H.annuus,is a popular oil-producing herb available worldwide nowadays.The plant is spread globally for foods and oils.It has different uses.The leaves,roots and flowers are used in the various system by the local people to treat swellings, snakebites, spider bites, malaria, rheumatic aches,soap,and candles[21-23].The selected oil-producing plant is locally available but not a single work was done on the antimicrobial and cytotoxic potential of the seeds and flowers,which is grown in Oman.In this context,the study was undertaken to evaluate the antimicrobial and cytotoxic potential of different polarities seed and flower extracts of H.annuus.The collected samples were processed according to standard procedure for washing,grinding,and extraction.The extraction was done by standard protocol with methanol and the solvent-free extract was dissolve in water and successively fractionation with a various solvent with increasing polarity.Those various polarities extracts were used for the estimation of antimicrobial and cytotoxic potential followed by standard procedure.

3.1.Extraction

The coarse samples of flowers and seeds of H.annuus were extracted with direct methanol by using a Soxhlet extraction method at 70°C for five days.After extraction,it was filtered and evaporated the methanol solvent by a rotary evaporator at 25°C until dryness.Then both the flowers(35 mg)and seeds(50 mg)extracts were dissolved in water(200 mL)by using a sonication.Both dissolved extracts were fractionated separately by using a separatory funnel and give the hexane,chloroform,ethyl acetate,butanol,and water extracts,respectively that are presented in Table 1.

3.2.Antimicrobial potential

The in vitro antibacterial potential of the seeds and flowers various polarities extracts of H.annuus were determined by the disc diffusion technique reported by Weli et al.[24] with modification.Five bacterial strains,which were three grams-positive and two grams-negative pathogenic bacterial strains.The pathogenic microorganism that culture which was collected from the Microbiology Laboratory at the Nizwa Hospital.In our present experiment,nutrient agar media was used for the culture of bacteria.Four concentrations of each extract of seeds and flowers were prepared by using the DMSO.Also, positive and negative control levofloxacin and DMSO were used.The positive control was prepared with the same solvent at the concentration of 1 mg/3 mL.The fractionated extracts from flowers and seeds give the significant inhibition against Gram-positive Staphylococcus,Streptococcus,and S.aureus and G(−)E.coli and Klebsiella at the concentration of 500,250,125 and 61.5 μg/mL.The inhibition of fractionated different polarities crude extracts at various concentrations were in the range of 0-20 mm (Table 2).Among the flower extracts, the height inhibition as antimicrobial potential was found in water extract and the lowest potential was in butanol extract followed by water ＞ethyl acetate ＞hexane ＞methanol ＞chloroform ＞butanol extracts.The maximum zone of inhibition 20 mm was obtained from ethyl acetate extract among the fractionated extracts at the concentration of 500 μg/mL against Staphylococcus bacterial strains.It was also given promising inhibition against gram-negative bacteria Klebsiella at a concentration500 μg/mL range of 18 mm.Similarly,the other bacteria also give good inhibition within the range of 0-15 mm at the concentration of 250,125 and 61.5 μg/mL(Table 2).The hexane extract from flowers gave the significant inhibition against all applied bacterial strains at all concentrations.Among the bacteria, the highest inhibition was obtained in hexane extract at 500 μg/mL against E.coli.However,the hexane extract did not show any potential against Streptococcus at the concentrations of 125 and 62.5 μg/mL.The methanol extract obtained from the flowers of H.annuus gave the potent inhibition against all gram(+and −)bacterial strains at all concentrations(Table 2).The inhibition ranges in between 0 and 15 nm compare to the positive control.The chloroform and water extracts at various concentrations also give significant potential against the applied pathogenic bacterial strains.Almost all the extracts showed a high zone of inhibition at all concentrations it could be due to antimicrobial guided chemical ingredient present in the methanol and it derived fractionated extracts.There are no reports available on the antimicrobial potential of the Omani H.annuus.Therefore,the obtained experimental results on antimicrobial potential were unable to compare with other reports due to the unavailability of data.However,the selected plant species in other countries showed similar results[29,30].

Table 1 Percentage of extracts from flowers and seeds of H.annuus.

Similarly,among the seed extracts,the highest potential was obtained in water extract and the lowest potential was in methanol extract followed by water ＞hexane ＞butanol ＞ethyl acetate ＞chloroform ＞methanol extract.The highest zone of inhibition 15 mm was obtained in chloroform extract at the concentration of 500 μg/mL against S.aureus.The other concentrations of chloroform extract showed moderate inhibition against the applied Staphylococcus,Streptococcus,S.aureus,E.coli and Klebsiella pathogenic bacteria strains.Not only that the water extract,but the other fractions derived from methanol extract gave the same moderate inhibition against the applied bacterial strains(Table 3).The range zone inhibition was 0-15 mm.The potential index of the various seed extract was calculated which was reported [31].The methanol and butanol extracts did not give any inhibition against the E.coli and Klebsiella.Almost,the several authors in other countries reported similar antimicrobial potential on H.annuus extracts[30,31].

3.3.Cytotoxic potential

Brine shrimp lethality bioassay was used to evaluate the cytotoxic potential of various polarities seeds and flower extracts of H.annuus which is described by Rehab and Hossain[24,32].In this present experiment,the DMSO solvent was used as a positive control.The seeds and flower extracts of various polarities at different concentrations 500,250,125,62.5,31.25 μg/mL were used.After incubation for the period of 24 h at ambient temperature,the survival nauplius in the test tube at different polarities and different concentration extracts was calculated using a magnifying glass.The determination of LC50values of the selected flower and seeds extracts was evaluated by using the help of the Probity Analysis Chart using computer EXCEL software(Fig.1).All the isolated fractions derived from methanol extract including methanol extract were tested for toxicity by using brine shrimp nauplii.All fractions of seeds and flower extracts of H.annuus gave cytotoxic potential (Fig.1).The results of cytotoxicity among the flower extracts against the brine shrimp method where the highest LC50against brine shrimp nauplii was found to be 31.25 μg/mL in water extract and the lowest LC50were 1345.86 μg/mL in chloroform extract followed by water ＞ethyl acetate ＞hexane ＞butanol ＞methanol ＞chloroform extract.Similarly, among the various polarities seeds extracts, the highest LC50against brine shrimp nauplii was showed to be 109.56 μg/mL in butanol extract and the lowest LC50was 1513.56 μg/mL in ethyl acetate extract followed by butanol ＞hexane ＞water ＞methanol ＞chloroform ＞ethyl acetate extract.According to the definition of LC50,the less LC50value means it has potent toxicity.That means,in the present experiment of flower samples, water extracts contained the highest toxic extract (less LC50value means high toxicity) and chloroform having the lowest active extract among the various flower extracts.Therefore,the water extract from theflower samples is the most potent toxic extract among the five prepared extracts from H.annuus.

Table 2 Antimicrobial activity of different flower crude extracts of H.annuus.

The cause of cytotoxic of the plant extract completely depends on its chemical constituents.The water extract of the flower was found to be the highest cytotoxic potential; it could be due to the phytochemicals.Similarly, the butanol extract showed the highest potential; it could be the toxic phytochemical present in the butanol extract.This study showed that the extracts from flowers and seeds having promising cytotoxicity could be incorporated as an anti-cancer drug apart from its antibacterial property.In our best knowledge,there was no reported data stating thebiopotential of the flowers and seeds of Omani H.annuus so far referring to the other parameters on remaining parts of the selected plant.The above studies on the various flowers and seeds extracts of H.annuus,it is decided that the plants may represent new sources of antimicrobials and anticancer agents,biologically active components that can create a logical base for the use of plants in modern medicine.These herbal preparations and prescriptions of plant sources,which are available in the global market,should be scientifically screened and then distributed properly.Based on experience and knowledge about the herbal preparation of natural sources of medicinal plants can be extended for future investigation into the field of pharmacology, pharmacognosy, chemistry, ethnobotany and other biological actions for drug discovery.

Table 3 Antimicrobial activity of different seeds crude extracts of H.annuus.

Fig.1.IC50 values of different flowers and seeds crude extracts.

4.Conclusion

Both the antimicrobial potential and cytotoxic potential of the prepared various polarities flowers and seeds extracts of H.annuus were measured by well-recognized bioassays.The antimicrobial potential of all flower and seed extracts showed signification extracts against the gram-positive and negative bacterial strains.Among the flower extracts,the highest potential was obtained in water extract and the lowest potential was in butanol extract.In the seed extracts,the highest potential was obtained in water extract and the lowest potential was in methanol extract.The cytotoxicity among the flower extracts against the brine shrimp method where the highest LC50against brine shrimp nauplii was found to be 31.25 μg/mL in water extract and the lowest LC50was 1345.86 μg/mL in chloroform extract.Similarly,various polarities of seed extracts,the highest LC50against brine shrimp nauplii was showed to be 109.56 μg/mL in butanol extract and the lowest LC50was 1513.56 μg/mL in ethyl acetate extract.However,the potent flower and seed extracts might be used as a safe medicine for the preparation of antibiotics and anticancer drugs.Therefore,further extensive phytochemical and pharmacological studies are needed for the confirmation of biological potential.It is also needed to isolate,separate,and identify antimicrobial and cytotoxic guided phytochemicals.Furthermore,in vivo pharmacological studies are needed for a better understanding of their mechanism of action.

Declaration of competing interest

The authors declare no conflicts of interest in relation to this work.

Acknowledgment

The authors are grateful to the University of Nizwa for the opportunity it has offered us to do this present work.We also thank Miss Nadiya Banioraba,the Lab technician for her assistance in finishing our project work.We also extend our appreciation to Mr.Musa Atanda Folami,English Language Instructor,Foundation Institute,University of Nizwa for his kind help with English corrections.