BIOLOGICAL ACTIVITIES OF SOME SELECTED NEPALESE MEDICINAL PLANTS AND ISOLATION OF CHEMICAL CONSTITUENTS FROM CALLICARPA MACROPHYLLA

Objective: The main objectives of this study was to analyze the phytochemicals, determine the total flavonoid content, brine shrimp toxicity, antibacterial activity, evaluate the antioxidant, antimicrobial, anti-diabetic activities of nine medicinal plants Callicarpamacrophylla, Bauhinia purpurea, Plumeriarubra, Girardiniadiversifolia, Acacia nilotica, Woodfordiafruticosa (Bark) Woodfordiafruticosa (flower), Terminaliaalata, and Premnabarbata. 
Methods: The cold percolation method was adopted for the extraction of secondary metabolites in methanol. The preliminary phytochemical analysis was performed by colour differentiation methods. The radical scavenging activity was evaluated by DPPH (2,2-diphenyl-1-picrylhydrazyl) method. The antidiabetic activity was performed by α-amylase enzyme inhibition activity. The chemical constituent was isolated by column chromatography from the biologically active plant fraction. 
Results: The phytochemical investigation has shown plants are the rich source of secondary metabolites as quinones, saponins, terpenoids and glycosides. Among the nine tested plants, Terminaliaatalia showed the highest radical scavenging activity 96.41±0.47 with IC50 value 6.353 µg/ml, followed by Girardiniadiversifolia 97.26±0.67 with IC50 value 11.52 µg/ml whereas ascorbic acid has 39.85 µg/ml as standard. Bauhinia purpurea showed significant inhibition to the α-amylase enzyme having inhibitory concentration IC50 17.05±13.00 SD in a dose-dependent manner. Woodfordiafruticosa demonstrated significant toxicity to A. salina with LC50 value of 457.08 µg/ml. Callicarpamacrophylla bark showed a potential inhibitory activity against the growth of Straphylococcusaureus as compared to standard chloramphenicol. Active plant extract of Callicarpamacrophylla was subjected for column chromatography. Conclusion: Out of nine plant samples Terminaliaatalia showed the highest radical scavenging activity. The plant extract of Bauhinia purpurea showed significant inhibition to the α-amylase enzyme inhibition. Woodfordiafruticosa demonstrated significant toxicity to A. salina, whereas Callicarpamacrophylla showed the potent antibacterial activity. The active plant extract was subjected for column chromatography and different fractions were collected in solvent polarity basis. 
Conclusion: The phytochemical investigations showed that plant extracts are the rich sources of secondary metabolites such as alkaloids, flavonoids, saponins, glycosides, polyphenols, coumarins and reducing sugars which showed they are supposed to be responsible for different biological activities. IC50 values showed the varied degree of antioxidant property of which Plumeriarubra and Acacia nilotica exhibit good antioxidant property with IC50 value close to the standard ascorbic acid.


INTRODUCTION
Nepal is rich in all three levels of biodiversity, namely species diversity, genetic diversity and habitat diversity. In Nepal, large number of medicinal plants are known to have medical values and peoples have been using since many years to cure specific diseases [1]. Nepal has been regarded as the natural showroom of biodiversity because of its geotopography which is reflected in its dramatic contrast of climatic condition, which in turn is reflected in floral and faunal variations. Such biodiversity has supported the live hood of people who live in remote areas of Nepal. These local people of a different ethnic group traditionally acquired a diversity of knowledge regarding the utilization of plant and animal resources for various purposes like food, medicine, clothing construction, dyes, ritual performances, energy, etc. About 80-90% people living in rural areas of Nepal depend directly or indirectly on the formal and informal system of traditional medicine involves the use of plant extracts [2]. Antioxidants are natural or synthetic substances that may prevent or delay oxidative cell damage in human beings. In humans, free radicals have been blamed, at least partially, for the development of several chronic ailments, for example, Alzheimer's disease, atherosclerosis, cancers and many others [3].

International Journal of Current Pharmaceutical Research
Oxidative stress is a pathological state in which reactive oxygen/nitrogen species (ROS/RNS) overwhelm antioxidative defense of the organism, leading to oxidative modification of lipids, proteins, DNA, tissue injury and accelerated cellular death [3]. Commercially available antioxidants are butylated hydroxytoluene (BHT), butylated hydroxyanisole (BHA) and tertiary butylated hydroxyquinone (TBHQ). But, these antioxidants have side effects and toxicity when taken in vivo. Hence, their use is being restricted and have an urgent need to find out safer and bioactive natural antioxidant [4,5]. Diabetes mellitus is a metabolic disorder characterized by loss of glucose homeostasis occurring due to defects in insulin secretion or insulin action resulting from impaired metabolism of glucose, lipids and proteins. Hyperglycemia, the primary clinical diagnosis of diabetes, is thought to be contribute to diabetic complications by altering vascular cellular metabolism in human body. Diabetes is a multifactorial diseases leading to several complications require a multiple therapeutic approach [6,25]. α-amylase is an enzyme that breaksα bonds of large polysaccharides, such as starch and glycogen yielding glucose and maltose [8,24]. It is the major form of amylase found in humans and other mammals [7]. α-amylase inhibitory agent is a protein family which inhibits mammalian α-amylases mainly by forming a stoichiometric complex with α-amylase [9].
An antimicrobial agent either kills microorganisms or inhibits their growth. Antimicrobial medicines can be grouped according to the effect caused by microorganisms in human body [10]. Today, numerous antimicrobial agents exist to treat a wide range of infections. The development of new anticancer and antimicrobial therapeutic agents is one of the fundamental goals in medicinal chemistry. One new strategy for the research on new anticancer and antimicrobial therapeutic agents has been the use of metalcontaining compounds. The antimicrobials should be selectively toxic to the pathogenic microbes but not toxic to the host tissues [10]. More ethnopharmacological studies have been performed in Nepal but these results are not well documented and explored. The peoples of different communities of Nepal have been using such medicinal plants for cure of simple to life threating diseases but the modes of preparation and administration of traditional herbal medicines are not well known. The evidences that show the relationship between pharmacological and phytochemical uses of plants are not well explored [13].
The most important part of this research is documenting the traditional knowledge and perform scientific validation of traditionally used different natural products, especially medicinal plants. Validation can be performed by different in vitro and in vivo experiments or by isolating the target secondary metabolites, which is useful for treating particular diseases or any health disorders [11,12]. Present study focused on the collection of nine medicinal plants, Callicarpamacrophylla, Bauhinia purpurea, Plumeriarubra, Girardiniadiversifolia, Acacia nilotica, Woodfordiafruticosa (bark), Woodfordiafruticosa (flower), Terminaliaatalia and Premnabarbata from Palpa district of Nepal based on ethnomedicinal and traditional uses of plants and to perform their scientific validation as the primary source of medicine curing different diseases. Based on their biological activities, one of the plant fraction was selected to isolate the chemical compound by column chromatography.

Sample preparation
The bark of Callicarpamacrophylla, Bauhinia purpurea, Plumeriarubra, Acacia nilotica, Woodfordiafruticosa, Terminaliaalata, Premnabarbata, and flower of Woodfordiafruticosa and the root of Girardiniadiversifolia were collected and washed with tap water to remove the contaminants. Then the collected plant parts were shade dried. The dried plant parts were grinded into powder form in electric grinder and stored in clean plastic bag at 4 °C until to perform different biological activities.

Extract preparation
The phytochemicals were extracted by cold percolation method using methanol as a solvent. Powdered plant parts (150 g) of mentioned plants were kept separately in the clean and dry conical flasks. Methanol (400 ml) was added to each nine different flask and kept for 72 h with frequent shaking. The mixtures were decanted and filtered with the help of cotton plug and thus obtained filtrates were concentrated with the help of rotatory evaporator by distillation at temperatures below 60 °C. The concentrated filtrates were kept in a beaker wrapping with aluminum foil containing small pores to facilitate the evaporation of the solvent. After complete evaporation of the solvent extracts were obtained. These plant extracts were stored at 4 °C until doing biological activities. Biological activities were performed after sudden extraction. Percentage yield for each plant extracts was calculated.

Phytochemical analysis
This method involves the selective and successive extraction of phytochemicals where the method adopted was primarily based on the standard procedure. The analysis of the presence of main groups of natural compounds in the different plant extracts was done by the color reaction using different specific reagents [16].

Antioxidant activity
This method is rapid, simple and inexpensive to measure antioxidant capacity involves the use of the free radical, 2,2diphenyl-1-picrylhydrazyl (DPPH). The ability of different plant extracts to scavenge DPPH free radicals was performed by adopting the standard protocol described by Jamuna et al. 2012 [17].
Different concentrations of test samples of 20, 40, 60, 80 and 100 µg/ml were made from stock solutions. Then 2 ml of each plant extracts were mixed with 2 ml of DPPH solution. The test tubes were shaken vigorously for the uniform mixing then the solutions was kept for 30 min in the dark at room temperature. After 30 min, absorbance was measured at 517 nm using a UV-visible spectrophotometer. Ascorbic acid of same concentrations was used as a standard.
The percentage of the DPPH free radical scavenging activity was calculated by using the equation, Where, A0= Absorbance of the control (DPPH solution+methanol), As The IC = Absorbance of test sample 50 indicated as the effective concentration of the sample that is required to scavenge 50% of the DPPH free radical. IC50

Antidiabetic activity (α-amylase inhibition assay)
values were calculated using the inhibition curve by plotting extract concentration versus the corresponding scavenging.
The antidiabetic activity of plant extracts was determined by using the α-amylase inhibition assay proposed by Kusano et al. 2011with few modification. The undigested starch due to enzyme inhibition was detected through the blue starch iodine complex at 630 nm [18].
1000 µg/ml of stock solution of different dry extracts were prepared by dissolving 17 mg dry mass of extract in 17 ml dimethyl sulphoxide (DMSO). This stock solution was further used to prepare 5 different concentrations of each extracts viz. 640 µg/ml, 320 µg/ml, 160 µg/ml, 80 µg/ml and 40 µg/ml. Substrate was prepared by dissolving 200 mg of starch in 25 ml of NaOH (0.4M) by heating at 100 °C for 5 min. After cooling, pH was adjusted to 7.0 and the final volume was made up to 100 ml using distilled water. 400 µl of substrate was pre-incubated with 200 µl of varying concentrations (640 µg/ml, 320 µg/ml, 160 µg/ml, 80 µg/ml and 40 µg/ml) of plant extracts and acarbose separately at 37 °C for 5 min. After this 200 µl of α-amylase solution was added to each of them and then again incubated for 15 min at 37 °C. After incubation the enzymatic reaction was quenched with 800 µl of HCl (0.1M). Then, 1000 µl of iodine reagent was added, and the absorbance was measured at 630 nm. Then experiment was carried out in triplicate. Percentage of enzyme inhibition was calculated by using formula, Where, Abs1 = absorbance of incubated mixture containing plant extract, starch and amylase, Abs2 = absorbance of incubated mixture containing plant extract and starch, Abs3 = absorbance of incubated mixture containing starch and α-amylase, Abs4 = absorbance of incubated solution containing starch only. Graph was plotted by taking the concentration on the x-axis and percentage inhibition on the y-axis. With the help of this graph, IC50 values of each samples were calculated. The species having the lowest IC50 was considered to have the best α-amylase inhibition property.

Qualitative screening and evaluation of an antibacterial activity
Sterile Muller-Hinton Agar (MHA) plates were dried to remove excess of moisture from the surface of the media. The agar plates for the essay were prepared by labeling them with the name of the bacteria and the name code of the disc. The inoculums of bacteria were transferred into petri disc containing solid nutrient media of agar using sterile swab. The plate was rotated through an angle of 60 °after each swabbing. The swab was passed around the edges of the agar surface. The inoculated plates were left to dry for minutes at room temperature with a lid closed. Four wells were made in each incubated media plates with the help of sterile cork borer no.6. So, the diameter of a well was 6 mm and labeled properly. Then 50 µl of the working solution of the plant extract, DMSO as negative control and 25 µl of ofloxacin as a positive control at the same time were loaded into the respective wells with the help of micropipette. The plates were then left for half an hour with the lid closed so that the extract diffused into media. The plates were incubated overnight at 37 °C. After 24 h of incubation, the plates were observed for the presence of inhibition of bacterial growth indicated by a clear zone around the wells. The size of the zone of inhibition was measured and the antibacterial activity expressed in terms of the average diameter of zone of inhibition in millimeters. The absence of zone of inhibition was interpreted as the absence of activity. The ZOI were measured with the help of a millimeter ruler and the mean was recorded [19].

Brine shrimp bioassay (Toxicity test)
The eggs of brine shrimp are readily available at low cost and they remain viable for years in the dry state. Upon being placed in a brine solution, the eggs hatch within 48 h providing large number of larvae (nauplii). It determines the LC50 value (S) (µg/ml) for the crude extract (s). Extracts having LC50values less than 1000 ppm (µg/ml) are considered as pharmacological active. Compounds/ extracts having LC50 LC values less than 1000 ppm (µg/ml) are considered as pharmacological active. The assay was carried out by adopting the standard protocol of Meyer et al. 1982 [20]. 50 If 'n' is the number of replicates (here three), 'x' is the log of constituents in mg/ml (log10, log100 and log1000 for three dose level respectively), y is prohibit for average survivor of all replicates.
value is the lethal concentration dose required to kill 50% of the shrimps. It can be determined as follows, Where,

From prohibit regression,
Where Y is constant

LC50
In the present work, brine shrimp bioassay of different plant extracts was carried out and the lethal concentration value was calculated.

Extraction and isolation of pure compounds
On the basis of biological activities, the plant extract of Callicarpamacrophylla was selected as an active sample for the isolation of compounds by chromatographic technique. Bark of C. macrophylla was dried and powdered. 150 g of powdered plant material was extracted with methanol by cold percolation. The solvent was filtered and evaporated in a rotatory evaporator to get methanol extract. The yield of the methanolic extract obtained was 15.56 g. The methanolic extract was then fractionated with different solvents such as hexane, dichloromethane, ethyl acetate and methanol-based on polarity.

Chromatographic separation
The hexane fraction weighing 8.01 g was adsorbed on 20 g silica gel and loaded on to a silica gel (120 g, Qualigens, and 60-120 mesh) packed column having an internal diameter of 3 cm with the adsorbent height 32 cm. The column was initially eluted with hexane and then the gradient of hexane in ethylacetate of increasing polarity and finally reported upto 100% ethyl acetate. Different fractions were collected and analyzed by thin-layer chromatography (TLC). Based on TLC report hexane fraction was selected for isolation of chemical constituents by column chromatography.

Yield percentage of plant extracts
Quantitative estimation of plant extracts showed different yield percentage shown in table 2. The plant sample Callicarpamacrophylla showed the highest yield percentage (10.37%), indicating the plant extract is the rich source of secondary metabolites. The plant extract of Acacia niloticca showed the lowest yield percentage indicating the extract is the poor source of secondary metabolites as phytoconstituents.

Phytochemical screening
The results of the phytochemical analysis is shown in table 3.
The degree of decolorization indicates the free radical scavenging potentials i.e. antioxidant potentials of the sample.
Percentage scavenging of the DPPH radical was gradually increased with the increase in the concentration of the methanolic plant extract from 20-100 µg/ml. The percentage inhibition of DPPH free radical of methanolic extract of bark of Callicarpamacrophylla and Terminaliaalata was found almost equal to the standard ascorbic acid taken whereas nearly equal to the bark of Acacia nilotica and flowers and barks of Woodfordiafruticosa. Graphical representations of DPPH assay of all the extracts is shown in fig. 1.
The linear regression of the percentage of radical scavenging versus concentration was used for the calculation of the concentration of each plant extract required for 50% inhibition of DPPH activity (IC50). The antioxidant potential has an inverse relation with IC50 value, lower the IC50 indicates high antioxidant potential. The IC50 values of the plant extracts along with the standard ascorbic acid is shown in table 4. Values are expressed as mean±SD with n=3 The inhibitory concentration of Terminaliaalata, Girardiniadiversifolia and Callicarpamacrophylla showed low IC50

Antibacterial activity
value with high antioxidant potential. These plant samples are the good sources of natural antioxidants. The rest of the plant extracts are moderate towards antioxidant activity with respect to the standard ascorbic acid. The antioxidant potential of plant sample was found comparable to the previously reported results [21,22]. The results perform the scientific validation to the plant extracts that have been using by the peoples since many years to cure simple and life threating diseases.
The diameter of zone of inhibition (ZOI) produced by plant extracts on particular bacteria was measured for the estimation of their antimicrobial activity. The methanolic extact of Plumeriarubra, Bauhinia purpurea, Premnabarbata, Woodfordiafruticosa and Terminaliaalata did not show any zone of inhibition at 10 mg/ml. Further, extracts of Callicarpamacrophylla, Girardiniadiversifoliaand Acacia nilotica were found not to be resistant against E. coli, whereas the same extract were found to be resistant against S. aureus. The extract of Callicarpamacrophylla, Girardiniadiversifolia and Acacia nilotica found active for the inhibition of the growth of S. aureus only whereas negative response towards E. coli. The Callicarpamacrophylla showed the highest ZOI (12 mm) against S. aureus. Terminaliaalata against E. coli showed 20 mm of ZOI.

α-amylase inhibition activity
The absorbance of different test samples was recorded by spectrophotometer. The graph was plotted concentration of plant extract against the percentage α-amylase inhibition, where acarbose was used as the positive control. The IC50 The comparisons of percentage α-amylase inhibition between different plant extracts and acarbose as standard are shown in the fig. 2.
values of each extracts were calculated with the help of plot.   The IC50 values of different plants extract along with standard acarbose were evaluated and found that the value ranges from 17.05 µg/ml to 4308.25 µg/ml. From the data the extract of Woodfordiafruticosa (flower) having IC50 value 366.52 µg/ml which is close to the standard acarbose with 361.01 µg/ml IC50

Brine shrimp bioassay (Toxicity test)
value. The plant extracts of Bauhinia purpurea, Woodfordiafruticosa (bark), Plumeriarubra,), and Acacia nilotica are found potent than the acarbose. The rest of the plant extacts showed poor inhibitory activity against the α-amylase inhibition activity. Previous research reported that the aqueous leaves extracts of P. Americana possess hypoglycemic activity. Similarly, different fractions of R. Ellipticus fruits were reported for its antidiabetic activity on alloxan-induced diabetes and glucose tolerance test in rats. The results showed the similarity in α-amylase inhibition activity as reported by the previous researchers [23].
The toxicity of different plant extracts were evaluated for their toxicity towards newly hatched Brine Shrimp Larvae (A. salina leach) adopting the protocol Mayer et al. 1982. In this study, the lethal concentration that kills 50% of the exposed population of A. salina (LC50 The degree of lethality was found to be directly proportional to the concentration of the extracts that is maximum mortalities of the brine shrimp larvae took place at the concentration of 1000 µg/ml and least mortalities were at 10 µg/ml. Those having LC ) values in µg/ml for different concentrations of plant extracts was determined and results obtained during these studies were recorded. 50 values less than 1000 µg/ml are supposed to be pharmacologically active. It is cleared that the plant extract of sample AB6

Isolation of compounds
was found toxic towards the brine shrimp larvae whereas rest of the plant extracts were found nontoxic. Although this method does not provide any adequate information regarding the mechanism of toxic action, it is a very useful method for the assessment of the toxic potential of various plant extracts. This method provides preliminary screening data that can be backed up by more specific bioassays once the active compounds have been isolated.
On the basis of antioxidant activity and anti-diabetic nature of Callicarpamacrophylla extract was selected to separate the chemical constituents by column chromatography. The hexane fraction of methanolic extract weighing 3.5 g was adsorbed on 20 g of silica gel to make a slurry and loaded on silica gel packed column. The column was eluted in increasing order of solvent polarity and different fractions were collected/examined by thin-layer chromatography. The results of TLC examination for different fractions collected after elution is shown in table 7.