Anti-Leishmanial Evaluation of Fraxinus xanthoxyloides (G. Don) DC. Collected from District Islamabad.
Summary: In this study, we have investigated phytochemical classes, antileishmanial and cytotoxic activity of Fraxinus xanthoxyloides (Oleaceae) leaves. Powder of F. xanthoxyloides leaves was extracted with methanol to obtain the crude extract (FXM) and the resultant was fractionated with solvents in escalating polarity; n-hexane (FXH), chloroform (FXC), ethyl acetate (FXE), n-butanol (FXB) and the residual aqueous (FXA) fraction. Quantitative estimation of terpenoids, coumarins, flavonoids, phenolics and tannins was conducted. Anti-leishmanial activity was performed against Leishmania tropica promastigote stage parasite while insecticidal activity was assessed through brine shrimps lethality assay. Our results showed the maximum concentration of terpenoids in FXC while the highest quantity of coumarins, flavonoids, phenolics and tannins was recorded in FXE. Presence of terpenoids was not detected in FXB and in FXA.
Among the extract/fractions, FXC exhibited the highest anti-leishmanial activity with LD50 of 15.23+-0.9 ug/ml to that of glucantime (LD50 = 5.6+-2.4 ug/ml) a reference drug. FXH exhibited the anti-leishmanial activity of LD50 = 40.68+-1.9 ug/ml followed by FXE (LD50 = 102.9+-3.1 ug/ml). Similarly potent insecticidal activity was recorded (LD50 = 28.15+-1.8 ug/ml) for FXC followed by FXH (LD50 = 67.59+-2.3 ug/ml). However, other fractions exhibited low level of anti-leishmanial and insecticidal activity. Correlation analysis exhibited a strong association (p 0.05) with the insecticidal activity. The coumarins established a medium association with the insecticidal activity.
Other chemical classes exhibited a moderate to low level of association with the anti-leishmanial and the insecticidal activity. On the basis of these results we can conclude that chloroform fraction of F. xanthoxyloides is a potential source for anti-leishmanial and insecticidal activities and further studies are required to isolate the active constituents.
Keywords: Fraxinus xanthoxyloides; Phytochemical; Anti-leishmanial; Cytotoxicity; Brine shrimp.
Extensive evaluation of medicinal plants and their folk uses has been carried out in last 40-50 years research. World Health Organization has nominated 252 drugs as "essential drugs" of which 11 % are derived from the natural products . Plant kingdom being an excellent source of easily isolated drugs has earned domination to herbal products being safer, cost effective and with reduced side effects than that of synthetic compounds .
The activities of plants extract against different infections may reside in the variety of different structures, essential oils, phenolics and aldehyde compounds . The secondary metabolites i.e. tannins, terpenoids, coumarins, alkaloids, phenolic compounds, flavonoids present in plants show variety of biological activities like antioxidant, cytotoxic as well as antiparasitic potential [3, 4].
Leishmaniasis, is a group of diseases the causative agent of which belong to several species of Leishmania exhibiting different forms of clinical symptoms. It is the largest occurring disease among the vector borne diseases after flariasis and malaria . WHO categorized leishmaniasis as a category 1 disease, i.e. uncontrollable and emerging . Three clinical types of leishmaniasis has been recognized; visceral, cutaneous and mucocutaneous leishmaniasis. Visceral leishmaniasis accounts 500,000 new cases each year in 61 countries and among these 90% cases belong to India, Bangladesh, Nepal, Sudan, and North Eastern Brazil .
In Pakistan leishmaniasis was first accounted in northern areas in 1960. In the beginning it was restricted to northern sphere but now it is widely spreading throughout the country . Both cutaneous and visceral leishmaniasis is the major threats to Pakistan despite of, mucocutaneous leishmaniasis which is rarely reported . Leishmania tropica is the causative agent of cutaneous leishmaniasis and is accompanied by long lasting ulcers on stratum cornium of skin tissue, which results in patient's disgrace and humiliation in society. Cutaneous leishmaniasis is a vector borne disease and is transmitted from person to person by a third party; sandfly. The infection is readily disseminated within a specific area depending upon several factors like population density, poor hygiene, improper sanitation and lack of protection from blood sucking insects like mosquito, flee and lice .
Issues of toxicity and resistance is therefore compelling us to ponder and work on a new therapy for this disease which is clinically more efficacious than currently available medication, have milder side effects and is cost effective. So scientists are working hard to develop such drugs from plant sources that are easily available and have low production cost.
Cancer is a leading cause of death all over the world, about 12 million people all over the world are diagnosed cancer each year and it kills more people than tuberculosis, malaria and AIDS collectively. Among various bioassays to assess antitumor activity brine shrimp lethality assay mostly used to screen the plant extracts/fractions or compounds, because of strong association of the human nasopharyngeal carcinoma with brine shrimp toxicity. It provides fundamental guidelines to isolate active compounds . This method is practiced quite often to assess possibility of anticancer activity of plant extracts and then develop new anticancer drugs. Already a number of cytotoxic and pesticidal agents are developed through this system. Screening of medicinal plants for phytochemicals and antiparasitic activities is important for finding the potential raw compounds for therapeutic use.
In our study we have selected Fraxinus xanthoxyloides for quantitative determination of secondary metabolites, anti-leishmanial and insecticidal activities.
The leaves of F. xanthoxyloides were collected in October, 2013 from the campus of Quaid-i-Azam University Islamabad, Pakistan. The plants were recognized by their local names and then validated by Rizwana Aleem Qureshi, Professor, Plant Sciences Department, Quaid-i-Azam.University Islamabad, Pakistan. Specimen (45679) was submitted to National Herbarium, Quaid-i- Azam.University, Islamabad.
After collection, plant samples were shade dried till the complete removal of moisture and samples were made to mesh sized powder by using plant grinder. Powder (2kg) of the sample was soaked in crude methanol (4L) for extraction for 72 h. All the samples were processed two times repeating above procedure. For the purpose of filtration, Whatman No. 1 filter was used and methanol was evaporated on a rotary evaporator at 40degC under reduced pressure. To sort the compounds in the crude extract with increasing polarity, crude extract (30g) was suspended in distilled water (250ml) and passed to liquid-liquid partition by using solvents as n-hexane (FXH), chloroform (FXC), ethyl acetate (FXE), n- butanol (FXB) and aqueous (FXA) fractions. Rotary evaporator was used to concentrate the fraction by evaporating the solvent under reduced pressure at 40degC .
Extracts and fractions were dried and then stored at 4degC for the investigation of phytochemical constituents, insecticidal and anti-leishmanial activities.
Quantitative Estimation of Secondary Metabolites
Determination of Terpenoids
An aliquot of 1.5ml of chloroform was mixed with 100ul of extract/fraction (10mg/ml). After 3 min of mixing 100ul of conc. sulphuric acid (H2SO4) was added and the assay tubes were incubated for 5 min. Reddish brown precipitate formed was dissolved in 1.5ml of 95% methanol. The spectrophotometer was adjusted to a 538 nm wavelength to measure the absorbance. The test was performed in at least triplicate and the total terpenoid content was expressed as milligrams of linalool equivalents per gram of the sample extract (mg LE/g) .
Determination of Coumarins
To quantify the coumarins, 500ul (10mg/ml) of the extract/fractions was transferred to a test tube followed by the addition of 2ml of distilled water and 500ul of 1% lead acetate. After thorough shaking, 7ml of distilled water was added to the reaction mixture. A volume of 2ml of the reaction mixture was mixed with 8ml of hydrochloric acid (1N) and allowed to stand at room temperature for 30 min and absorbance was recorded at 320 nm. The test was performed in triplicate and the total coumarin content was expressed as milligrams of coumarin equivalents (1,2-benzopyrone) per gram of the sample extract (mg CE/g) .
Determination of Total Flavonoids
Flavonoid content was determined following Park et al.  with some modifications. In a 10ml test tube, 500ul (10mg/ml) of crude extract and fractions, 3.4ml of 30% methanol, 150ul of 5% sodium nitrite (NaNO2) and 300ul of 10% aluminium chloride hexahydrate (AlCl3.6H2O) were added and mixed. After 5 min, 1ml of 1M sodium hydroxide (NaOH) was added and allowed to stand for 5 min.
The absorbance was measured at 510 nm by using UV/VIS-DAD spectrophotometer. The standard curve for total flavonoids was made using rutin standard solution (6.25-100 ug/ml) under the same procedure as above. The total flavonoids were expressed as milligrams of rutin equivalents per gram (mg RE/g) of dried sample.
Determination of Total Phenolics
The total phenolics were determined in the extract/fractions by the spectrophotometric method . In brief, a 300ul of the test sample was transferred to test tube and 2.25ml of Folin-Ciocalteu reagent (previously diluted with distilled water by 10 fold) was added and then mixed. The resulting mixture was allowed to stand at room temperature for 5 min. Then 2.25ml of 6% (w/v) sodium carbonate was added to above mixture. The resulting mixture was placed for 90 min at room temperature and absorbance was taken at 725 nm by using UV/VIS- DAD spectrophotometer. The standard curve for total phenolics was made using gallic acid standard solution (6.25-100ug/ml) under the same procedure as above. The total phenolics were expressed as milligrams of gallic acid equivalents per gram of dried sample.
Determination of Tannins
For the quantitative determination of tannins polyvinyl polypyrrolidone (PVPP) assisted quantification of tannins was done . A test tube containing 100mg of PVPP was mixed with 1ml of distilled water followed by 1ml of test sample (10mg/ml). After 4 h of reservation at 4degC the reaction mixture was thoroughly mixed and centrifuged (2000 x g for 10 min at room temperature) to get bilayer mixture. PVPP causes the precipitation of tannins while supernatant contains the phenolics other than tannins. Then phenolic contents were determined as above  in the supernatant as non-tannin phenolics. Tannins are then calculated by this formula:
Tannin = Total phenolics - non-tannin phenolics
Stock solution for anti-leishmanial assay was prepared by dissolving 5mg/ml of each plant extract and derived fractions in 1ml of DMSO. Stock solutions were further diluted serially (1000, 700, 500, 50, 5, 0.5 and 0.05ug/ml) using DMSO to obtain the appropriate concentrations. Samples were filtered by using a 0.45um syringe filter. Leishmania tropica KWH23 was previously isolated from a patient in Peshawar, Pakistan and was characterized (data not shown). The promastigotes form of the Leishmania were grown in M199 medium with 10% Fetal calf serum (FCS), HEPES buffer, streptomycin and penicillin. Log phase promastigotes at 1 x 106/100ul were used for the entire assay. About 90ul of 199 media, 50ul of Leishmania tropica KWH23 log phase culture and 10ul of each plant dilution was dispensed to different wells of micro-titter plate. Here, DMSO (10ul) was used as a negative control, while glucantime as positive control.
Afterwards, micro-titter plate was incubated at 24degC for 72 h. After incubation about 15ul of each dilution was pipetted on a neubauer counting chamber and were counted under a microscope .
To estimate the toxicity of the plant extracts and their fractions, brine shrimp lethality assay was used. For the hatching of brine-shrimps (Artemia salina) eggs, at an ambient temperature of 23 +- 1degC artificial sea water (3.8g sea salt/L) was used . Three different concentrations of each extract (1000, 100 and 10ug/ml) were made, taken from 10mg/ml stock solution in methanol. Methanol was evaporated before transferring shrimps to the vials. After 24 h, the hatched shrimps were shifted to the vials filled with 5ml of artificial sea water (10 shrimps per vial) along with samples. As a positive control, from stock solution of tricaine methanesulfonate (1mg/ml) serial dilutions were made in the range of 0.05 ug/ml - 100ug/ml in DMSO. The number of the shrimps that survived the sample environment was counted after 24 h.
Characterization of extract/fractions for cytotoxicity was demonstrated according to You et al.  by the sulforhodamine B assay. Briefly, 190 ul of RAW 264.7 (ATCC-TIB-71) cells with a density of 5x104 cells/ml were seeded in 96-well plate having 10 ul of the test sample (final concentration of 20 ug/ml) in DMSO (10%) and PBS, and incubated for 72 h at 37 oC in CO2 incubator. After incubation 50 ul of 20% TCA was added to terminate the reaction. Cells were washed, dried and stained with 0.4% of acetic acid for 30 min at room temperature. Cells were washed four times with acetic acid and dried overnight. Bound dye was solubilized in 200 ul of 10 mM of Tris base (pH:10) on a gyratory shaker for 10 min. Absorbance of each treatment was recorded at 515 nm through a micro- plate reader.
A zero-day control was performed in each case following addition of equal quantity of cells in sixteen wells, with subsequent incubation for 30 min at 37 oC and was processed as mentioned earlier. Cell survival percentage was calculated for each test sample.
GraphPad Prism software version 5.0 (2007) was used to calculate LD50 values of each sample for brine shrimp toxicity assay and anti-leishmanial activity. Nonlinear regression test was used to determine R2, and LD50 values. Correlation between the LD50 value of the anti-leishmanial and insecticidal activity with the chemical class was established by using the Statistix 8.1.
Results and Discussion
In order to cure different diseases, the residents of Pakistan belonging to rural areas are dependent on the indigenous system of medicine. On the basis of conventional use of plants, we carried out the in vitro biological screening of crude methanol extract and its derived fractions of F. xanthoxyloides leaves. It is well acknowledged, that the medicinal value of plants is due to the presence of bioactive phyto-components . Hence the study of plant constituents is helpful to explain the traditional uses of plants. In this investigation crude methanol extract and fractions of F. xanthoxyloides were investigated for the quantitative determination of terpenoids, coumarins, flavonoids, tannins and phenolics (Table-1). Medicinal plants have prime importance to community health and various pharmacological effects are produced due to the presence of plant secondary metabolites. Secondary metabolites like terpenoids, tannins and flavonoids have various beneficial roles to cure various human ailments.
The amount of phytochemicals which were found in extract/fractions of F. xanthoxyloides was determined by the standard procedures. Table-1 depicted the terpenoids content in extract/fractions of F. xanthoxyloides which were estimated as milligrams of linalool equivalents (LE) per gram of sample. In our study the highest terpenoid content was found in FXC (100.41mg/g) followed by FXM (73.16mg/g) > FXH (51.41mg/g) > FXE (17.75mg/g) while in FXB and FXA terpenoids were not present. Terpenoids have earned a reputation as anti- inflammatory, analgesic, hepatoprotective, antipyretic, cardiotonic, general tonic and sedative agent in many Asian countries [17,18]. Recent studies added more belief that terpenoids are not only capable of above mentioned pharmacological properties but also have antioxidant, antiviral, antiangiogenic, antimicrobial, anti allergic, antipyretic and spasmolytic aptitude [19- 21].
An admiring number of triterpenoids have been reported to possess selective cytotoxic ability against a range of cancer cells without any harmful effect to the normal cells [22, 23].
Total coumarin contents were estimated in terms of 1,2-benzopyrone equivalents (BPE) which are presented in Table-1. Coumarin contents were high in FXE (60.20mg BPE/g sample) as compared to other fractions and the lowest amount was observed in FXH (0.40mg BPE/g sample). Coumarins are lactones of O-hydroxy-cinnamic acid derived from trans-cinnamic acid via oxidation- reduction and isomerization to produce 1,2- benzopyrone. Coumarins can be categorized into simple coumarins, pyranocoumarins, furanocoumarins, chromones and dimeric coumarins. Coumarins have antioxidant, antispasmodic, spasmolytic, anti-HIV, hypolipidemic, vasodilating and hypotensive capacities .
Total flavonoid content (TFC) was calculated as rutin equivalent (RE) and was varied noticeably in the extract/fractions of F. xanthoxyloides (Table-1). This study showed the highest TFC in FXE (44.04mg RE/g sample), followed by FXB (43.37mg RE/g sample) > FXM (41.70mg RE/g sample) > FXC (34.76mg RE/g sample) > FXA (9.70mg RE/g sample) > FXH (2.82mg RE/g sample). According to reports the phenolics can be scrutinized into several classes, of which flavonoids being the major one have potential antioxidant capabilities . Naturally occurring secondary metabolites in plants like flavonoids are quite beneficial to human health. Studies have affirmed the antibacterial, anti-inflammatory, antiviral, anticancer and anti-allergic aptitude of flavonoids derivatives [24, 25].
Table-1: Quantitative screening of phytochemicals in F. xanthoxyloides.
###Extract/###Total terpenoids###Total coumarins###Total flavonoids###Total phenolics###Total tannins
###Fraction###mg/g dry sample###mg BPE/g dry sample###mg RE/g dry sample###mg GAE/g dry sample###mg GAE/g dry sample
###FXB###0e###28.00c+-2.2###43.37a +-4.1###241.53c +-2.9###46.60b+-2.4
###FXA###0e###11.80e+-1.4###9.7cc +- 0.9###206.87d +-3.2###33.40c+-1.7
Table-2: Anti-leishmanial screening of various fractions of F. xanthoxyloides.
Total phenolic contents (TPC) of all the extract/fractions were calculated in terms of gallic acid equivalents (TAE) as shown in Table-1. The present study showed the TPC in the subjected plant as: FXM (262.74mg TAE/g sample) > FXE (256.36mg GAE/g sample) > FXB (241.53mg GAE/g sample > FXA (206.87mg GAE/g sample) > FXC (203.08mg GAE/g sample) > FXH (202.22mg GAE/g sample). Because of improving quality, nutritional value of foods and impeding oxidative degradation of lipids, phenolics rich plant materials have earned a reputation . Phenolics, the phenylalanine and tyrosine derivatives are considered as secondary metabolites which occur universally in plants and are diversified . Table-1 presented the total tannin contents. We observed that tannins were high in FXE (81.4mg GAE/g sample) and FXC (79.2mg GAE/g sample) and the lowest amount was observed in FXH (1.2mg GAE/g sample) which was almost negligible.
Wani et al.  screened the methanol, ethyl acetate and hexane extracts of Hyptis spicigera plant for qualitative and quantitative analysis of phytochemicals through standard procedures. Quantitative analysis showed the presence of terpenoids (16.10%), phenolics (20.75%), alkaloids (7.55%), flavonoids (8.82%) and saponins (6.23%). Methanol extract of Gentiana kurroo Royle (Gentianaceae) roots was screened by  and declared this important and omnipresent medicinal plant of Kashmir Himalaya to possess several bioactive metabolites and nociceptive activity. Tannins, saponins, cardiac glycosides, alkaloids, terpenes, carbohydrates, flavonoids and phenolics were found to be present on phytochemical evaluation.
Table-2 displays the LD50 values for anti- leishmanial activities of the crude methanol extract and its derived fraction of F. xanthoxyloides. It was recorded that chloroform fraction exhibited the best anti-leishmanial activity (LD50=15.23+-0.9ug/ml, R2=0.999) whereas the least effective was the aqueous fraction which have displayed the highest LD50=2314+-3.6ug/ml, R2=0.999 value. The LD50 value recorded for the standard drug glucantime was 5.6+-2.4ug/ml, R2=0.999. Anti-leishmanial activities of various plants have been reported in earlier studies. Leaf extract of Anisomeles malabarica and Ricinus communis exhibited good anti-leishmanial activity (LD50 = 126+-19.70 and 184+-39.33ug/ml), respectively . Similarly Shah et al.  reported the anti-leishmanial activity of Jurenia dolomiaea roots against Leishmania tropica KWH23 promastigotes.
The methanol extract of J. dolomiaea roots exhibited LD50 = 10.9+-1.1ug/ml while its ethyl acetate fraction manifested LD50 = 5.3+-0.2ug/ml) anti-leishmanial activity against L. tropica.
Anti-leishmanial activity of F. xanthoxyloides has been reported for the first time in this study. The treatments currently in use as prime therapy for leishmaniasis include meglumine antimoniate (Glucantime), in addition to pentavalent antimonials sodium stibogluconate (Pentostam), but they are harmful to some extent, require prolong parenteral administration courses and have powerful side effects . In some cases, pentamidine as well as amphotericin B are used as second line treatment which may also have lethal effects . The results of our study revealed pharmacological activity against L. tropica by F. xanthoxyloides and our data suggests that the chloroform fraction has the potential use for leishmaniasis. Further studies are required to investigate the active compounds responsible for anti-leishmanial activity and their in vivo application to sought out their appropriate concentration against Leishmania in infected experimental organisms.
It would be helpful in designing new medicines which are biologically more active and cost-effective and having minimum side effects.
Insecticidal screening using brine shrimps bioassays was carried out to provide important preliminary data to screen plant extracts for their anti-cancerous potential. Insecticidal effect of crude extract and various fractions of F. xanthoxyloides were studied for insecticidal activity through BSLA under controlled conditions. Direct relation was found between brine shrimps lethality and concentration of different extracts/fractions (Table-3). LD50 is measure of therapeutic index of a substance/extract. LD50 was the lowest concentration of test sample at which deaths of half of the shrimps took place. Data of present study indicated that the order of LD50 for F. xanthoxyloides was in the following order i.e. FXC > FXH > FXM > FXB > FXA > FXE. In case of insecticidal studies chloroform fraction of F. xanthoxyloides showed the best activity, (LD50 = 28.15+-1.8ug/ml).
The association between the LD50 value of the anti-leishmanial and insecticidal activity with chemical classes is given in Table-4. The results indicated significant (p 90% survival was determined for the tested samples. In view of these results it can be established that plant is non toxic at 20ug/ml to these cells.
Table-5: Cytotoxicity assay for F. xanthoxyloides extract/fractions used in the in vitro model.
It can be concluded that, F. xanthoxyloides is an asset for the field of ethnopharmacology as it is used traditionally in various regions of Pakistan to solve different health related problems. Therefore, there is a need to isolate bioactive compounds from this plants which may be potential source for their anti-leishmanial and insecticidal activities.
Competing Interest: The authors declare no competing interest.
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|Publication:||Journal of the Chemical Society of Pakistan|
|Date:||Oct 31, 2016|
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