AAE= Ascorbic acid equivalent; GAE= Gallic acid equivalent; TAE= Tannic acid equivalent; LC₅₀= Median lethal concentrations; OD=Optical density; ME: Methanol fraction; PETSF: Petroleum ether portion; N-Hex: n-hexane portion; CH: Chloroform portion; SK: Streptokinase

Phytomedicine refers to the use of any plant part for medicinal purposes [1]. Estimates of 25% of currently used prescribed drugs are chemically altered natural products and another 25% comes directly from the arena of herbal medicine [2]. In addition, evidences drawn from the studies of Arvigo et al (1993) showed an estimate of 119 chemical substances were obtained from 90 different plant species. In 74% cases, an active substance was isolated from a plant which had been used in the traditional system of medicine, revealing the primary importance medicinal plants in the healthcare system [2]. This is due to the fact that, plants contain various secondary metabolites serving as the source of novel therapeutic compound conferring improvement of human health with controlled adverse effects. Various medicinal products like anti-microbial agents, cardiotonic agents, Central nervous system (CNS) stimulants, nutraceuticals, sweeteners, animal feed, etc, are derived from the natural source [3-5], natural product can also have the potentiality of anticancer agent [6].Thus the present study aims at the preliminary phytochemical screening and the evaluation of diverse pharmacological activities of the methanol extract of Murdannianudiflora, Tradescantiapallida and Piper nigrum as well as their organic soluble partitionates (Figures 1 and 2).

Figure 1: Whole Plant of Doveweed (Murdannianudiflora).

Figure 2: Whole Plant of Purple Heart (Tradescantiapallida)

The plants employed for the study had some notable compound that have elucidated significant therapeutic activity such as anthrocyanidins isolated from Tradescantia pallid was investigated as potential food pigment as well as various flavonoid compound isolated from the seeds of Piper nigrum such as piperine, Limonen, Sabinene in abundance as well as b-cryophyllene, Piperolein B, Dehydropipernonaline, Piperlongumininehas been investigated for its therapeutic role as major antioxidant compounds.

Majority of the diseases/disorders are mainly linked to oxidative stress due to free radicals. Antioxidants can interfere with the oxidation process by reacting with free radicals, chelating, catalytic metals, and also by acting as oxygen scavengers [7,8]. Therefore, a number of reports on the isolation and testing of plant derived antioxidants have been described during the past decade [7-10]. Brine shrimp lethality bioassay serves as a preliminary tool to assess the cytotoxicity as well as the anti-tumor activity of bioactive natural products [11].

Pain is a symptom as an outcome of injury and disease conditions whose management is always critical and vital i.e., to provide comfort to the patients, consequently, derived analgesic drugs are becoming significant day by day [12].

The erythrocyte membrane resembles to lysosomal membrane and as such, the effect of drugs on the stabilization of erythrocyte could be extrapolated to the stabilization of lysosomal membrane [13]. As a result, membrane stabilizers that interfere in the release and/ or the action of mediators like histamine, serotonin, prostaglandins, leukotrienes etc., could reduce the inflammation significantly [14]. Thrombolytic drugs, such as urokinase and streptokinase, play a crucial role in the management of coagulation disorders e.g., cerebral venous sinus thrombosis (CVST) i.e., a disorder often accompanied by significant morbidity and mortality [15].

In view of the above, the present study strives to highlight the antioxidant potential, cytotoxicity and analgesic activity of M. nudiflora and T. pallida as well as the membrane stabilizing, thrombolytic and cytotoxic abilities of P. nigrum.

Plant materials

Murdannianudiflora: M. nudiflora (L) Brenan, commonly termed as Doveweed, is an annual herb being one of the only two recognized species of the genus Murdannia under the family Commelinaceae [16]. Widely distributed in wet place, the plant is mainly found in low elevated areas as well as in the tropical and subtropical regions, with rarely can be seen among grass [17,18].Traditionally used in the cure of asthma, leprosy, stomach complaints, piles, the plants has been also imparted its use as an astringent. The root paste mixed with goat milk has been prescribed orally for the cure of asthma, however, the whole plant paste in combination with a common salt has been used to cure the affected areas of leprosy [19]. Evidences shows the presence of significant group of phytochemicals such tannins, flavonoids and alkaloids while the ethanolic extract has shown significant level of analgesic effect using hot plate test (P<0.05) where morphine sulphate has been used as standard [20].

Tradescantia pallida: T. pallida (commonly known as Purple Heart), widely distributed from Canada to northern Argentinais generally commercialized as ornamental houseplant. In traditional systems of medicine, the plant has shown its potential as antiinflammatory and anti-toxic supplement in addition to its ability of improving blood circulation obtained from the evidence in Taiwanese traditional medicine [21]. In terms of its environmental value, the plant has been found out to be an effective removal of volatile organic pollutant from the air [22].

Extensive studies over its antioxidant potential of the leaf extract revealed a total flavonoid content of 10.6 ± 4mg GAE (Gallic Acid Equivalent)/100mg while an estimate of total tannin content was 13.6 ± 2.1mg TAE (Tannic Acid Equivalent)/100mg [23]. The studies further showed the free radical scavenging capacity of the leaf extract to be 103.1 ± 36.9mg AAE (Ascorbic Acid Equivalent)/100 g [23]. Isolation of chemicals from the plant extract has led to discovery of two anthocyanidins, a notable flavonoid group namely, cyaniding-g 3, 73’-triglucoside with 3 molecules of ferulic acid and an extra terminal glucose while the other lacking the terminal glucose in studies done by Shi et al, 1995 for its potential use as food pigment [24]. In terms of its antimicrobial potential, the leaf extract has shown various degree of zone of inhibition of 5-10 mm against various gram-positive and gram-negative micro-organisms while a zone of inhibition of more than 10 mm has been showed against Staphylococcus epidermis [23]. In terms of environmental values, the plant has shown its ability to survive in presence of chromium concentration raging 5-20 mg of L(01)Cr(VI) in hydrophobic environment while the plant has shown indication of accumulation of heavy metals like Chromium as per studies conducted by Sinha et al, 2014 [25].

Piper nigrum: The seed of Piper nigrum Linn a common spice known as white pepper, is considered as the king of spice. It is native to India and tropical countries such has Indonesia (Java in particular) as well as Malaysia, Madagascar and different parts of South East Asia [26] which showed rich values in terms of therapeutic properties available throughout the year [27]. Traditional system of medicine has given potential leads of pepper in epilepsy and snake bite. While Syrian books of medicine found in 5th Century AD has shown pepper as medicine for illnesses like constipation, dysentery, earache, gangrene, heart disease, herniation, hoarness, digestive problems, insect bites, insomnia, pain at the joints, liver, lung disorders, tooth decay and toothache while the root has shown its potential in the form of ghees, powder, enemas and balks for the remedy of abdominal tumor [28].

Presence of high degree of polyphenol with addition to its potential in free radical scavenging has led to the emphasis of its antioxidant value as well as its significant role in the prevention of various oxidative stress and its associated disorders which however has not been confirmed with evidence on in vivo studies [29].

The spice has also showed to affect the growth rate of Streprococcus mutans which is a strong indicative of its antimicrobial properties [30].

Collection of the plant materials

Doveweed was collected from Savar, Dhaka whilePurple Heart was collected from the National arboriculture Department, Ministry of Public Works, and Government of the People’s Republic of Bangladesh. A White Pepper seed was collected from a local market. After collection, the plant materials were identified and confirmed by the National Herbarium, Ministry of Environment and Forests, Government of the People’s Republic of Bangladesh, Mirpur, Dhaka (DACB Accession No: 40297, 40299) (Figures 3 and 4).

Figure 3: Distrubution Map of Doveweed [cabi.org].

Figure 4: Distrubution map of Purple Heart [cabi.org].

Maceration

The study materials were subjected to the process of shed drying for a period of a month with minimal exposure to direct sunlight and the materials had undergone grinding after cutting down into small pieces.

The plant materials were first dissolved into 2 lit of methanol while the White Pepper seeds were dissolved individually under 500ml of methanol, chloroform and n-hexane (Active Fine Chemicals, Bangladesh). The contents were kept for a weeks, occasionally stirred and shaken. The dissolved materials were filtrated firstly through cotton and afterwards through Whatman No.1 Filter Paper (Extra Large Size) which was concentrated to obtain crude extracts of different solvent of the study materials involving rotary evaporator (Heidolph Rota-Vap 1500) at an elevated temperature range of 35-50ºc involving a rotation speed of 80-100 rpm.

Phytochemical identification

Phytochemical Screening was carried out to for the qualitative identification of major groups of phytochemicals found in the plant extract. Around 30mg of the crude plant extract was dissolved in 30ml of distilled water with a few drop of DMSO (Fischer Chemicals Limited) , added to facilitate dissolution of the crude extract. Phytochemical identification tests of carbohydrates, tannins, saponins, flavonoids, alkaloids, protein was carried out as per the standard procedures [31] (Figures 5 and 6).

Figure 5: Herbarium Sheet of Murdannianudiflora (DACB Accession No.: 40297).

Figure 6: Herbarium Sheet of Tradescantiapallida (DACB Accession No.:40299).

Evaluation of cytotoxic potential

Preliminary cytotoxic potential of the study materials were evaluated using Brine Shrimp Lethality Bioassay [32]. Using simulated conditions for 48 hours, Brine shrimp was hatched in artificial sea water. The mother concentration of the crude extract were prepared by dissolving in DMSO (Di-methyl sulphoxide) from which serial dilution has taken place by adding equal amount of dissolved sample and DMSO. The dissolved samples of various concentrations were added to pre-marked vial of simulated seawater having 10 nauplii in each of them. Vincristine sulphate was used for the evaluation of positive control of cytotoxicity while negative control of the study was conducted using 5ml of simulated sea water containing 10 nauplii where DMSO was added. The vials were left for 24 hours and the surviving nauplii were counted to evaluate cytotoxicity of the samples by visual observations.

The mortality data were analyzed using probit analysis and linear regression method which were expressed in terms of median lethal concentration (LC₅₀) value obtained by utilizing regression equation (Figure 7-13).

Figure 7: Cytotoxic Assay of Murdannianudiflora Crude Methanolic Extract.

Figure 8: Cytotoxic Assay of Tradescantiapallida Crude Methanolic Extract.

Figure 9: Cytotoxic Assay of Chloroform Extract of Piper nigrum.

Figure 10: Cyotoxic Assay of Methanolic Extract of Piper nigrum.

Figure 11: Cytotoxic Assay of n-hexane Extract of Piper nigrum.

Figure 12: Cytotoxic Assay of Petroleum Ether Extract of Piper nigrum.

Figure 13: Cytotoxic Assay of Vincristine Sulphate (Positive Control).

Evaluation of antioxidant potential

Antioxidant potential of the plant extracts were evaluated using DPPH (2,2-diphenyl-1-picrylhydrazyl) Free Radical Scavenging Method in order to determine the free radical scavenging ability and to have a quantitative measurement of the overall antioxidant potential of the sample [33]. Ascorbic Acid was used as positive control while methanol contributed as negative control of the study.

The mother solution was prepared by dissolving the plant material in methanol and subsequent serial dilution has taken place by dissolving the sample in equal amount of methanol. 1.8 ml of methanol was added to each of the pre-marked tubes of both samples at various concentration and positive control to obtain a final solution of 2 ml and was mixed thoroughly. 2 ml of DPPH was added to the pre-marked tubes and shaken well and was incubated in dark conditions for 30 m. Absorbance of the sample and control solutions was measured at 517nm by UV Spectrophotometer.

DPPH Radical Scavenging Activity was determined using the equation (% inhibition)=[{(A₀-A_s)/A₀}x 100], where A₀=Absorbance of the blank, A_s=Absorbance of the sample/standard

The equivalent value was determined using the equation: % of FRS_(sample)/% of FRS _(AA) x100 and IC₅₀ values were determined using linear regression and probit analysis in Microsoft Excel 2007.

In-vivo analgesic activity

Analgesic potential was tested using acetic acid induced writhing method. Study was conducted in two experimental test groups (Dose: 100 mg/kg and 200 mg/kg of body weight) for the crude plant extract while control group received oral saline (0.9% NaCl). Dichlofenac Na (25 mg/kg of body weight) was taken as standard for the study.

Test materials were orally administered using long needle with ball shaped end. Acetic acid (0.7%) was administered intra-peritonially to each of the testing animals after 30 minutes of oral administration of the sample. The counting of writhing was started after an interval of 5 m continued till 15 m allowing a time space of 10 m for each groups. Similar sets of conditions were applied for the control and standard groups as well (Figure 14).

Figure 14: Evaluation of DPPH Free Scavenging Power of plant extract (MN=MurdanniaNudiflora, TP= Tradescantiapallida, AA=Ascorbic Acid).

Comparison studies were conducted using percentage of inhibition calculated using the following equation: % of inhibition of writhing = (1-Mean writhing _(sample)/Mean writhing _(control)) x100.

Significance analysis of the study was conducted using t-test analysis (p<0.05) in IBM SPSS 17.0.

Evaluation of the membrane stabilizing activity

Whole blood from human under standard condition was collected and clotting was prevented using EDTA (Ethylene diamine tetra acetic acid). The blood sample was washed thrice with isotonic solution (154 mM NaCl) in 10 mM sodium phosphate buffer of pH 7.4 via centrifuge action for 10 m at 3000g to collect the stock erythrocyte suspension.

To conduct hypotonic solution-induced haemolysis, test sample consisted of stock erythrocyte suspension (0.50ml) with 5ml of hypotonic solution (50 mM NaCl) in 10mM sodium phosphate buffer containing the different crude extract (1.0 mg/ml). Acetyl Salicylic acid (0.10 mg/ml) was used as reference standard which has undergone similar set of experimental preparation. Incubation of the mixtures was done at room temperature for 10minutes, followed by centrifugation at 3000 g for 10 m and measurement of absorbance of the supernatant at 540nm. Percentage of inhibition was calculated using the following equation: % inhibition of haemolysis=100x{(OD₁-OD₂)/OD₁}, where OD₁= Optical density of hypotonic-buffered saline solution (control) and OD₂=Optical density of test sample in hypotonic solution (Figure 15).

Figure 15: Percentage of inhibition of writhing by different groups exposed to acetic acid induced writhing.

On the other hand, to evaluate membrane stabilizing potential using heat induced haemolysis, aliquot of 5ml containing 1 mg/ml of different extract of the sample dissolved in isotonic solution was put into two duplicate sets of centrifuge tube [31]. Same amount of vehicle was added to another tube as control. 30μl of erythrocyte suspension was added to each tube and gentle inversion was applied to mix properly. Two different sets of incubation was applied, one pair of the tubes was under 54ºC for 20 minutes while the other pair was maintained at 0-5ºC in an ice bath. Centrifugation at 1300 g of the reaction mixture was allowed for 3 m and absorbance of the supernatant was measured at 540 nm.

Percentage of inhibition or acceleration was calculated using the following equation: % inhibition of haemolysis=100x[1-(OD₂-OD₁)/ OD₃-OD₁]

Where, OD₁= Test sample unheated, OD₂=Heated test sample and OD₃=Control sample heated.

Evaluation of thrombolytic activity

Dry Crude Extract (100 mg) was suspended in 10ml of distilled to be kept overnight after which the soluble supernatant was decanted and filtered while commercially available streptokinase of 15,00,000 International Unit (IU) was collected and mixed properly with 5ml distilled water which was kept as a stock from which 100μl was used for in-vitro thrombolysis.

Whole blood was drawn from human healthy volunteer, without having a history of taking oral contraceptive or anticoagulant therapy from which 1ml of blood was transferred to previously weighed micro centrifuge tube allowed to form clots.

Aliquots of venous blood drawn from healthy volunteers (5 ml) were distribute five different pre weighed sterile micro centrifuge tube to be incubated at 37ºC for 45 minutes followed by removal of serum without disturbing the clot completely and further weighing the tubes to determine clot weight.

100μl of aqueous solutions of different crude extracts along was added separately. 100μl of streptokinase and 100μl of distilled water were taken as positive control and negative control, respectively, and were added to the control tubes separately. The tubes were then incubated at 37ºC for 90 minutes and visual observation of clot lysis has been conducted. Following incubation, the removal of released fluid has taken place and its subsequent weight was taken to observe the weight difference upon clot disruption. Percentage of clot lysis can be determined using the following equation:

% of clot lysis=(weight of released clot/ clot weight)x100.

Phytochemical screening

Phytochemical Screening of the plant materials confirmed the presence of alkaloids notable for its action against inflammation and pain, tannins significant for imparting anti-microbial and anti-tumor activity, compounds like saponins playing important role for its antihepatotoxic, anti-ulcerogenic, anti-carcinogenic activity as well as polyphenols like flavonoids for imparting significant antioxidant action that would combat against diseases related to oxidative stress as well as imparting its anti-bacterial action. Carbohydrate test showed positive results for M. nudiflora in the event of Fehling’s test which is an indication of presence of reducing sugar (Table 1). However, no protein molecules have been identified.

Table 1: Phytochemical Evaluation of Murdannianudiflora.

Evaluation of cytotoxicity

Poor degree of cytotoxicity has been showed by M. nudiflora and T. pallidain comparison to the positive control employed for the study. Taking LC₅₀ value into consideration, M. nudiflorahas a LC₅₀ value of 158.638μg/ml , while T. pallida has LC₅₀value of 833.85μg/ml in comparison to the standard drug having LC₅₀ value of 0.5926μg/ml. Identification of cytotoxic glycosphingolipid 2,β-O-D-glucopyranosyl- 2(2`-hydroxy-Z-6-enecosamide) sphingsonie from ethanolic extract of a herb of the similar genus named Murdannia loriform draws to the inference on the diversity of the notable secondary metabolite of the plant species belonging to same genus. This leads to another degree of potentiality that the plant extracts would be safe if ingested at high level indicative of its value as food supplement.

On the other hand, the seeds of Piper nigum have showed relatively high degree of cytotoxicity under the influence of various solvent. It was observed that the petroleum ether extract had the highest degree of cytotoxicity (LC₅₀ value=3.524μg/ml), closest to that of the standard drug used, while n-hexane extract had LC₅₀ value of 7.8101μg/ml, which is also indicative of high degree of cytotoxicity potential (Table 2). Similar trend of LC₅₀ values was found in studies conducted in the same procedure using the similar sets of solvent extract of dried leaves of Moringa oleifera where LC₅₀ values of petroleum ether extract was found out to lower than the LC₅₀ values of n-hexane extract [34]. The difference in biological activity between the pet-ether and the n-hexane soluble fractions may be attributed to the different sets of chemical compounds isolated from the partitionates. While steroidal compounds like stigmasterol were isolated from petroleum ether partitionate [35], n-hexane fraction yielded polyphenols and flavonoids [36].

Table 2: LC₅₀ values of the plant extracts and the standard.

Evaluation of antioxidant activity

After spectroscopic analysis, it was found out that the plant extract possesseda good degree of free radical scavenging capacity. M. nudiflora showed FRS capacity of 95.76 ± 1.34mg/100g of Ascorbic Acid while that of T. pallida was somewhat similar having FRS value of 97.15 ± 0.96mg/100g of Ascorbic Acid (Table 3).

Table 3: DPPH Free Radical Scavenging of Plant Extract Equivalent to Ascrobic Acid per 100g.

Comparing the IC₅₀ value of plant extracts in comparison to standard used showed good degree of antioxidant potential by M. nudiflora having an IC₅₀ value of 1.51x10-10μg/ml, while T.pallida possessed an IC₅₀ value of 5.48x10-10μg/ml.Ascorbic Acid showed IC₅₀ value of 4.209x10-13μg/ml (Table 4).

Table 4: IC₅₀ values of the plant extract and standard.

The results showed by the plant extracts in terms of free radical scavenging capacity as well as IC₅₀ values gives us a good indicative of the rich degree antioxidant potential the plant extracts possess in addition to the presence of high flavonoid content.

Analgesic potential evaluation

Analgesic potential of the crude plant extracts of M. nudiflora and T. pallida was evaluated using acetic acid induced writhing method and was found out to be statistically significant (p<0.001 and p<0.05, respectively) in comparison to control group.

M. nudiflora exhibited maximum inhibition of writhing at a dose of 200 mg/kg of body weight, having an inhibition of writhing of 85.67% which is higher than the standard utilized, while 100 mg/kg of body weight dose showed inhibition of writhing of 59.47%.

On the other hand, T. pallida inhibited writhing by 54.55% at a 200 mg/kg of body weight, while the dose of 100mg/kg of body weight showed 36.41% inhibition of writhing. The inhibitions of writhing at a dose of 200 mg/kg of body weight of T. pallida and for both the selected doses of M. nudiflora were found out to be statistically significant (Table 5).

Table 5: Evaluation of analgesic potential using acetic acid induced writhing method.

Presence of high degree of alkaloids obtained in phytochemical studies of the plant extract in relation to its potential of inhibition of writhing gives us potential lead of obtaining sources of new generation of analgesics. Since the etiology of pain generation is highly dependent upon the generation of prostaglandin and leukotriene derivatives, the results obtained from the study gives us potential directives in the discovery of analgesic products from natural sources highly acceptable taking the risks and adverse effects involved in administration of synthetic NSAIDs into consideration.

Membrane stabilizing capacity evaluation

In terms of hypotonic solution induced haemolysis, chloroform extract of white pepper exhibited an inhibition of 61.27%, while acetyl salicylic acid (standard) possessed an inhibitory capacity of 71.9%, which is an indicative of its high degree ofanti-inflammatory property (Table 6).

Table 6: Hypotonic induced haemolysis inhibition by different extracts of seeds of Piper nigrum.

In terms of heat-induced haemolysis, all extracts of different solvent showed high degree of membrane stabilizing activity in comparison to the standards used(acetyl salicylic acid, % of inhibition=57.42%). The highest degree of inhibition of heat-induced haemolysis of the seeds of P. nigrum was revealed by the chloroform extract (96.61%), while methanol extract (86.76%), petroleum ether extract (79.34%) and n-hexane extract (94.01) also possessed high degree of membrane stabilizing potential (Table 7) (Figure 16).

Table 7: Heat induced haemolysis inhibition by different extracts of seeds of Piper nigrum.

Figure 16: Hypotonic Solution Induced Haemolysis of Different Extracts of Piper nigrum.

Evaluation of thrombolytic activity of the seeds of P. nigrum upon dissolving in different solvents was assessed in comparison to thrombolytic drugs like Streptokinase (30,000 IU) with respect to its percentage of lysis (76.06%). It was found out that all the portions exhibited low degree of thrombolytic activity in comparison to the standard. The maximum thrombolytic activity was showed by methanol extract (30.74%) while the lowest was showed by n-Hexane portion (10.60%) (Table 8) (Figures 17 and 18).

Table 8: Thrombolytic activity of P. nigrumseed extracts.

Figure 17: Heat Induced Haemolysis of Different Extracts of Piper nigrum.

Figure 18: Thrombolytic Activity of Different Extracts of Piper nigrum.

In the world of advanced medical discovery where the compatibility of synthetic product is an issue in the illustration of therapeutic benefit with the added concern of toxicity and side effects, scientific justification of the natural product in therapeutic activity would serve as a basis for the newer avenues of complementary and alternative medicine to disease management as natural product minimizes the issues concerning compatibility of medication with human body. Therefore, this study aimed at identifying new and novel potential bioactive compounds based on the presence of notable phytochemicals and comparative evaluation on major bio-activity of various extract, thus providing the basis of entering into the world of therapeutically active natural compounds having the potentiality of providing safe alternative to the adverse effects of synthetic products in disease management. This comparison of various bio-activities of different plants and spices of Bangladesh would allow us in making a comparative justification on potential areas of further studies in order to validate the scientific evidence concretely. The study has led us to the scientific understanding of the rich antioxidant potential of M. nudiflora and T. pallida, high degree of cytotoxicity in various extracts of P. nigrum seeds as well as statistically significant inhibition of writhing shown by both the plants have opened the avenue of scientific research on the discovery of noble compound that contributes to the therapeutic effect mentioned in the study. In addition, presence of phytochemicals would allow the study on the plants on its anti-microbial activity, anti-carcinogenic activity, and hepatoprotective activities and so on. The results obtained from the bioactivity evaluation would also lead us to the evaluation of various fractions of crude extracts to obtain pure therapeutically significant compounds that impart significant bioactivities which contribute to the betterment of global healthcare.