Chemicals
Methanol, n-hexane, carbon tetrachloride, chloroform, ethyl acetate, and other chemicals used for the extraction, solvent–solvent partitioning of plant materials, in vitro, and in vivo pharmacological tests were laboratory grade (Merck, Germany).
Collection and identification of the plant
The matured plant leaves and stem barks were collected in August 2018 with the help of a famous local traditional healer. Then it was identified by a renowned taxonomist under the herbarium no-sr20385.
Preparation of crude extracts
Plant materials (leaves and stem barks) were washed, chopped into small pieces, and semi-shed sun-dried for seven days. After drying, the plant materials were powdered with a mechanical grinder. Powder portions of the leaves (1.36 kg) and stem barks (493 g) of M. dispermus were soaked in 7.29 L and 2.60 L of methanol, respectively. After 13 days of occasional shaking, the solution was filtered and the filtrate was concentrated by evaporation method under reduced pressure at the temperature below 50 °C by using a rotary evaporator (Stuart, UK). The weight of the crude methanol extracts of M. dispermus leaves and stem barks was 28.50 gm. and 7.66 gm., respectively. The percentage (%) yield of the extract was calculated using the following equation [14]:
$$\% {\text{ Yield of extract}} = \frac{{\text{Weight of extrcated material}}}{{\text{Weight of crude powder}}}$$
The percentage of yield of crude methanol extracts of M. dispermus leaves and stem barks was 2.09% and 1.55%, respectively.
Solvent–solvent partitioning
Crude methanol extracts of M. dispermus leaves were undergone solvent–solvent partitioning according to the protocol designed by Kupchan and Tsou and modified version of Wagenen et al. by using the solvents of n-hexane, carbon tetrachloride, chloroform, and ethyl acetate consecutively [24, 25].
Qualitative phytochemical screening
The preliminary phytochemical screening was done for evaluating the qualitative detection of terpenoids, flavonoids, saponins, phenol and tannins, phlobatannins, steroids, anthraquinones, alkaloids, glycosides, cardiac glycosides, resins, carbohydrates, proteins, fat and oil, and coumarin by using standard procedure [26,27,28]. The color intensity or the precipitate formation was used as analytical responses to these qualitative tests.
Experimental animals
Male Swiss Albino mice weighing approximately 20–30 g were used for experimental purposes. They were placed in standard propylene cages and familiarized under the controlled conditions (room temperature of 25 ± 2 °C, relative humidity of 60%-70% for 14 days) and operated with a 12 h light/dark cycle with food pellets. The mice were provided with a nutritionally adequate diet and drinking water ad libitum throughout the study. Diethyl ether anesthesia was employed on mice to euthanize. All the mice were sacrificed at the end of each experiment by using diethyl ether anesthesia. All segments of this report adhere to the ARRIVE Guidelines for reporting animal research. All experiments have been examined and approved by the ethical committee under approval no-cc98056.
Acute toxicity study
An acute toxicity study was conducted following the previously described method [29]. Each group comprises five Swiss albino mice and they fasted overnight before extract administration. Each group of animals was administered oral doses of 1000, 2000, 3000, and 4000 mg/kg of body weight of each of the extracts. After the administration of plant extract, they were restrained from food for further 3–4 h. Each animal was observed for the first 30 min, then for the first 24 h, and thereafter for 3 days. The mice were observed for any changes in the skin, fur, eyes, mucous membrane, respiration rate, circulatory rate, the central and autonomic nervous system at least once a day. The effective dose would be one-tenth of the median lethal dose (LD50).
Experimental design for in vivo testing
During the evaluation of analgesic activity, 16 groups of mice were used for each investigation and five mice were selected for each group. Group (I) was treated as control (1% tween-80 10 ml/kg), Group (II) was for standard (Diclofenac sodium 50 mg/kg used in acetic acid writhing study, and morphine sulfate 10 mg/kg served as the standard in both tail immersion and formalin-induced paw licking method) and others group were used for the administration of crude methanol extract of M. dispermus stem bark and leaves and its different extracted fractions at the dose of 200 and 400 mg/kg. For brewer’s yeast-induced antipyretic study, sixteen groups of mice were selected, numbered, five mice were assigned to each group. Group (I) was treated as control (1% tween-80 10 ml/kg), Group (II) was for standard Paracetamol (150 mg/kg) and other groups were used for the administration of crude methanol extract of M. dispermus stem bark and leaves and its different extracted fractions at the dose of 200 and 400 mg/kg. After each experimental period, all the treated mice were sacrificed using diethyl ether anesthesia.
In vitro studies of Macropanax dispermus extracts
Evaluation of thrombolytic activity
Blood specimen
Venous blood samples were drawn from 30 male and female healthy volunteers (age 18–26 years) who have no recent history of oral contraceptive and anticoagulant therapy. Ten blood samples were used for each concentration of each plant extracts. After that, 6 ml of venous blood were drawn from each volunteer. Blood was collected and preserved by an expert senior medical technologist.
A consent form was filed up for each volunteer for future reference.
Study design
Experiments for clot lysis were carried out as reported earlier [30]. About 500 μl of blood was taken into each pre-weighed Eppendorf tube and allowed to incubate at 37 °C for 45 min. After clot formation, fluid was completely released from each Eppendorf tubes and the clot weight was determined by subtracting the weight of the clot containing tube from the weight of the tube alone following the equation below:
$${\text{Weight of Clot}} = {\text{ Weight of Clot Filled Eppendorf}} - {\text{Weight of Empty Eppendorf}}$$
As standard, 100 μl of Streptokinase (SK) and, as a negative non-thrombolytic control, 100 μl of distilled water (DW) along with 100 μl of each sample of different concentrations (10 mg/ml, 8 mg/ml, 6 mg/ml, 3 mg/ml, 1.5 mg/ml) were separately added to the Eppendorf tubes. Then the Eppendorf tubes were incubated at 37 °C for 90 min. After incubation, the released fluid was discarded and tubes were again weighed to observe the difference in weight after clot disruption. Finally, the percentage (%) of clot lysis was determined as follows:
$${\text{\% Clot Lysis}} = \, \frac{{{\text{Weight of Clot Before Lysis}} - {\text{Weight of Clot Lysis }}}}{{\text{Weight of Clot Before Lysis}}} \times 100$$
Evaluation of cytotoxic activity
Preparation of seawater
Exactly 38 gm sea salt (without iodine) was dissolved in 1 L of distilled water and filtered off to get a clear solution. The pH of the seawater was maintained between 8.0–8.5 by applying 1 N NaOH solution [31].
Hatching of brine shrimp
Artemia salina leach (brine shrimp eggs) was collected from pet shops in Chittagong, used as the test organism. Seawater was taken in the small tank and shrimp eggs were added to the seawater in the tank. After 2 days (48 h) of hatching, the eggs would be matured as nauplii. The oxygen was supplied constantly through the oxygen pump during hatching time. The hatched shrimps were attracted to the light (phototaxis) and so nauplii free from eggshell was collected from the illuminated part of the tank. The nauplii were taken from the fish tank by a pipette.
Study design
Five milliliters of each of the plant extract solutions of different concentrations (1000, 500, 250, 125, 62.5 µg/ml) was added to 5 ml of seawater containing 10 nauplii. After 24 h, the Petri dishes were inspected using a magnifying glass against a black background and the number of survived nauplii in each was counted. The mortality endpoint of this bioassay was determined as the absence of controlled forward motion during 30 s of observation. From these data, the percentage (%) of the mortality of the brine shrimp nauplii was calculated for each concentration from the following equation [32]:
$${\text{\% Mortality}} = \frac{{N{\text{d}}}}{N} \times 100$$
Here Nd = Number of dead nauplii, N = Number of nauplii taken.
Determination of median lethal concentration (LC50)
The LC50 value represented the concentration of the extract that produced death in half of the brine shrimp nauplii after a certain exposure time and was determined by the linear regression method from plotting % of mortality against the correspondent concentration of the extracts. An approximate linear correlation was observed when concentration versus the percentage of mortality was plotted on the graph paper and the concentration–response data were transformed into a straight line utilizing a trend line fit linear regression analysis (Microsoft Excel 2007). The LC50 values were derived from the best-fit line obtained.
In vivo studies of Macropanax dispermus extracts
Evaluation of analgesic activity
Acetic acid-induced writhing method
This method was an analgesic behavioral observation assessment method that demonstrated a noxious stimulation in mice. This study was carried out using the method of Koster as modified by Dambisya and Lee [33, 34]. Fifteen minutes after administration of standard and 30 min after administration of the extract, 0.7% glacial acetic acid (10 ml/kg) was injected intraperitoneally (IP) to all the mice to induce pain characterized by abdominal constrictions or writhes. 5 min later, each mouse of all groups was observed to count the number of writhes for 20 min carefully and recorded. After each observation period, all the treated mice were euthanized using diethyl ether anesthesia. The percentage inhibition against abdominal writhing was used to assess the degree of analgesia and was calculated using the formula:
$${\text{\% of Pain Inhibition}} = \frac{{N_{\text{c}} - N_{\text{t}}}}{{N_{\text{c}}}}$$
Here Nc = number of writhings in the control group, and Nt = number of writhings in the treatment group.
Tail immersion method
This was a thermal method that was performed to evaluate the central analgesic property of the investigated extracts. The method described by Di Stasi et al. [35] was used for this experiment. Before 30 min of treatment about 2–3 cm of the tail of each of the mice was dipped into a water bath containing warm water maintained at a temperature of 50 ± 1 °C and the time taken for the mice to withdraw its tail from the warm water was recorded. The animals, which showed a flicking response within 3–5 s, were selected for the study. A cut-off period of 15 s was determined to avoid damage to the tail. After baseline, the treated mice were tested at 30, 60, 90, and 120 min after drug administration [36]. While measurements were being made, animals were briefly immobilized by wrapping them gently. After each observation period, all the treated mice were euthanized using diethyl ether anesthesia. The percentage of the Maximal Possible Effect (% MPE) was calculated using the following equation [37]:
$${\text{\% of MPE}} = \, \frac{{{\text{Post-drug Latency}} - {\text{Pre-drug Latency}}}}{{{\text{Cut off time}} - {\text{Pre-drug Latency}}}} \times 100$$
The percentage of time elongation was calculated from the following equation [38]:
$${\text{\% of Elongation of Latency}} = \, \frac{{{\text{Latency of Treatment}} - {\text{ Latency of Control}}}}{{\text{Latency of Treatment}}} \times 100$$
Formalin-induced paw licking method
This was a persistent-pain model that was used to assess both central and peripheral analgesic effects of the investigated extracts. This analgesic assay was done using a previously described method [39]. After sixty minutes of administration of control, standard, and investigated extracts, 20 µl of 1% formalin solution was injected through the subplantar route into the right hind paw of each mouse. The time (in seconds) spent licking or biting the injected paw indicated pain and was recorded. After subplantar injection of formalin, the responses of the mice were noticed for the first 5 min (early or neurogenic phase) and 15–30 min (late or inflammatory phase). After each observation period, all the treated mice were euthanized using diethyl ether anesthesia. The percentage (%) of pain inhibition was calculated using the following formula:
$${\text{\% Inhibition of Pain}} = \, \frac{{{\text{Reaction Time (Control)}} - {\text{Reaction Time (Treatment)}}}}{{\text{Reaction Time (Control)}}} \times 100$$
Evaluation of antipyretic activity
The antipyretic effect was assessed by using the brewer’s yeast-induced pyrexia method described by Adams et al. [40]. Before experimentation, the rectal temperature of mice was recorded by a digital thermometer. Hyperthermia was induced in mice by subcutaneous injection of 15% aqueous suspension of brewer’s yeast (10 ml/kg) in the back below the nape of the mice and the injected site was massaged to spread. Pre-drug temperatures were taken 24 h after the yeast injection to determine the pyretic response of yeast. Animals with 1 °F or more elevation in body temperature were used. The temperatures were recorded at 1, 2, 3, and 4 h intervals after the drug treatment. After each observation period, all the treated mice were euthanized using diethyl ether anesthesia. The percentage (%) reduction of rectal temperature could be calculated by the following formula [41]:
$${\text{\% Reduction of rectal temperature}} = \frac{B - C}{{B - A}} \times 100$$
Here A = Normal body temperature, B = Rectal temperature at 24 h after yeast administration, and C = Rectal temperature after drug administration at a different time interval.
Statistical analysis
All the data were expressed as mean ± SEM (Standard error of Mean). The results were analyzed statistically by one-way ANOVA followed by post hoc Dunnett's test using statistical software “Statistical Package for Social Science” (SPSS, Version 16.0, IBM Corporation, NY). Results below *p < 0.05, **p < 0.01, and ***p < 0.001 were considered statistically significant compared to control.
