"MORUS RUBRA L: “Phytochemical Screening, Anti- Bacterial, Antioxidant and Anti-Hyperglycemic Activity Determination"

Abbreviations: MR: Morus Rubra Linn; MIC: Minimum Inhibitory Concentration; MH: MuellerHinton; MBC: Minimum Bactericidal Concentration; SEM: Standard Error of the Mean; TSA: Tryptic Soy Agar Morus rubra L (MR), commonly known as red mulberry and is used as anti-phlogistic, diuretic, dysentery, expectorant and antidiabetic in the folk medicine. It belongs to the Moraceae family consists of a number of edible plants. The purpose of this study is to evaluate the phytochemical constituents, antimicrobial and anti-hyperglycaemic activity of ethanolic maceration and Soxhlet fruit extracts of Morus rubra MR. Fruit extracts were prepared using cold maceration and Soxhlet extraction methods with 95% ethanol. Preliminary phytochemical analysis for the important bioactive constituents from both Soxhlet and maceration ethanolic extracts were carried out. The results of phytochemical analysis revealed the presence of carbohydrates, monosaccharides, reducing sugar, hexose sugar, galactose, amino acid, protein, neutral lipids, saturated fatty acids, terpenoid, glycosides included anthraquinones and coumarin glycosides, alkaloid, phenolic compounds such as flavonoids, tannins and the absence of polysaccharides, pentose sugar, saponins, cardiac and cyanogenic glycosides, free fatty acid, steroids as well as volatile oil in red mulberry. The antibacterial activity of the ethanolic Soxhlet extract was determined by using broth tube dilution method to find out the relative MIC and MBC. For antimicrobial screening, Morica Rubra Soxhlet extract had no inhibitory or bactericidal effect against E. coli S. aureus. However, it showed antibacterial activity against S. aureus with equivalent MIC and MBC identified at the concentration of 500 mg/ml, 250 mg/ml and 125 mg/ml.


Introduction
Morus rubra Linn (MR), generally known as red mulberry belong to the Moraceae, mulberry family. It is originated from the eastern part of the United States of America and is now universally cultivated and naturalized in the areas of China, Korea and Japan.
Edible fruits that are reddishly maturing to dark purple or black in colour. The fruits are soft, juicy and aggregate with a sweet taste and a hint of sourness, which is more evident in the less mature fruits.
In addition, red mulberry sap was used by several tribes to treat ringworm while the stem bark is used as purgative and vermifuge.
The fruits have also been utilized to treat diabetes, hypertension, anaemia and arthritis in traditional medicine, particularly in Chinese medicine [1] (Figures 1-4). It is also demonstrated that Morus rubra (MR) fruit leads to control over hyperglycaemia and may be a good antimicrobial agent against gram negative bacterial infections. The first documented use of mulberry fruits was described in the mid-1500s by De Soto expedition, who discovered consuming these dried fruits [2]. Diabetes is a heterogeneous disease affecting almost 6% of the world population. It is characterized by hyperglycemia and insulin resistance or a combination of both of these factors.      (Miljkovic et al., 2014).

Antioxidant Assay
Antioxidant activity of ethanolic extract of MR is studied using DPPH method. The DPPH is a stable free-radical which has a colour of purple, as the antioxidants react with the DPPH, the DPPH will be converted into non-radical DPPH-H form. Besides that, the colour of the DPPH will be decolorized from purple to light yellow colour. The degree of decolorization indicates the potency of the plant extracts in scavenging the free radicals. 0.1M of methanolic DPPH was freshly prepared by dissolving 3.94mg of DPPH crystalline powder in 100ml of methanol and kept in a clean beaker. Extracts (0.2 ml) at different concentrations (50-1000 μg/ml) was mixed with 0.8 ml of tris hydrochloric acid (HCl) buffer (100 mM; pH 7.4). One millilitre DPPH (500 mM in 1.0 ml ethanol or methanol) solution was added to the mixture. The mixture was shaken vigorously and incubated for 30 min at room temperature. The absorbance of the resulting solution was measured spectrophotometrically at 518 nm (Model UV 1800, Shimadzu, Japan). BHT is used a standard. All the determinations were carried out in triplicate. The following formula was used for the antioxidant activity determination [9]. DPPH Radical Scavenging Activity (%) = −

Antibacterial Assay
The 10 mg/ml stock solution of ethanolic extract of MR is prepared using 0.5% DMSO (prepared using distilled water). A minimum of 5 days of acclimatization period was allowed before the animals are used in the experiment. The animals were fed with water and normal rodent pellet diet ad libitum. Diabetes mellitus will be induced in overnight-fasted rats by administration of single intraperitoneal (I.P.) injection of freshly prepared streptozotocin (STZ) with a dose of 60 mg/kg/mL [12]. To prevent the STZ-induced hypoglycaemia, rats administered with 10% dextrose solution after 24 h of STZ administration for next 24 h. Induction of diabetes was verified after 72 h by measuring blood glucose level with strips using glucometer and the animals allowed 14 days for the stabilization of blood glucose level [13]. On day 14, animals having a blood glucose level higher than 220 mg/dL were considered diabetic and included in the experiments. Diabetic animals were randomly divided into five groups (Group II-VI) as follows:

Statistical Analysis
The value in the antidiabetic activity was expressed as mean ± Standard Error of the Mean (SEM). The data was analysed using one-way analysis of variance (ANOVA), followed by Tukey's posthoc test. P < 0.05 will be considered as statistically significant.

Results
Phytochemical analysis showed the presence of carbohydrates,

Antimicrobial Screening
The The obtained result from the MIC test showed that turbidity was observed in all the assay tubes that tested against the S. aureus.
Therefore, three tubes with the least turbidity were selected and subjected for MBC.

Preparation of Tryptic Soy Agar Plate
Tryptic soy broth or trypticase soy broth (frequently abbreviated as TSB) is used as a culture broth to grow aerobic bacteria. It is a complex, general purpose medium that is routinely used to grow certain pathogenic bacteria, which tend to have high nutritional requirements (i.e., they are fastidious). Its agar counterpart is Tryptic Soy Agar (TSA). One of the components of Tryptic soy broth is Phytoene which is an enzymatic digest of soybean meal). TSB is frequently used in commercial diagnostics in conjunction with the additive sodium thioglycolate which promotes growth of anaerobes. Tryptic Soy Agar was only applied for the Bacillus subtilis out of the 4 chosen bacterial strains. 20g of tryptic soy agar powder was weighed and transferred into 500ml of distilled water. The conical flask was subjected to sterilization with the use of autoclave for 2 hours at the temperature of 121˚C.
In the biosafety cabinet, the freshly prepared sterilized tryptic soy agar was poured into the sterilized Petri plates. The inoculation loop was used to eliminate the bubble formed on the surface of the tryptic soy agar before solidification occurs. Approximately, 25ml of the tryptic soy agar was poured into the sterilized Petri plates, these plates were allowed to cool down and solidified in the biosafety cabinet. These plates were incubated in an upside-down position at the temperature of 37˚C and were taken out during the agar well diffusion test.

Agar Well Diffusion
Before the initiation of the agar well diffusion test, the biosafety cabinet was sterilized with 70% alcohol and the UV light was

Serial Dilution of Minimum Inhibitory Concentration (MIC) Assay
Before starting the MIC assay, the biosafety cabinet was sanitized with 70% alcohol and the UV light was switched on for 10 minutes.
2.5 g of plant extract was measured using an analytical balance and

Antioxidant screening
The MR were cleanly washed and air-dried at room temperature for 1 week to prevent deterioration. MR were passed through a sieve  The mixtures were shaken and labelled accordingly. The test tubes were allowed to keep in the dark for 30 minutes. The absorbance was measured at 518nm using a UV spectrophotometer.

Preparation of Sample:
The steps in preparation of DPPH solution were repeated in the preparation of the sample, in which the BHT was changed to the plant extract. The absorbance was measured at 518nm using a UV spectrophotometer.

Preparation of Blank:
The blank solution was prepared by mixing 3ml of 0.1mM DPPH reagent and 2.5ml of methanol, the solution was allowed to stand in the dark for 30 minutes. The absorbance was measured at 518nm using a UV spectrophotometer.
The values of absorbance of standard and sample obtained were then utilized in plotting the graph of UV absorbance against concentration of the sample and standard.

Antibacterial Assay Sterility Test of Ethanolic MR Extract
Before In the biosafety cabinet, the freshly prepared sterilized nutrient agar was poured into the sterilized Petri plates. The inoculation loop was used to eliminate the bubble formed on the surface of the nutrient agar before solidification occurs. Approximately, 25ml of the nutrient agar was poured into the sterilized Petri plates, these plates were allowed to cool down and solidified in the biosafety cabinet. These plates were incubated in an upside-down position at the temperature of 37˚C and were taken out during the agar well diffusion test.

Preparation of Tryptic Soy Agar Plate: Tryptic soy broth or
Trypticase soy broth (frequently abbreviated as TSB) is used as a culture broth to grow aerobic bacteria. It is a complex, general purpose medium that is routinely used to grow certain pathogenic bacteria, which tend to have high nutritional requirements (i.e., they are fastidious). Its agar counterpart is tryptic soy agar (TSA).

Determination of Total Phenolic Content (TPC):
In this research, the antioxidant potential of the MR was determined by using DPPH free radicals scavenging method and BHT was used as a standard. The DPPH is a stable free-radical which has a colour of purple, as the antioxidants react with the DPPH, the DPPH will be converted into nonradical DPPH-H form. Besides that, the colour of the DPPH will be decolorized from purple to light yellow colour.
The degree of decolorization indicates the potency of the plant extracts in scavenging the free radicals.  Table 8 and Graph 1 is drawn, respectively. The concentration, absorbance and the corresponding percentage of scavenging of BHT for DPPH Free Radical Scavenging Assay is shown in Table 9.
The concentration, absorbance and the corresponding percentage of scavenging of ethanolic MR extract is placed Table 10 while Graph of percentage scavenging against the standard concentration is drawn in Graph 2.    Graph 2: Graph of percentage scavenging against the standard concentration.
Graph 3: Graph of percentage scavenging against the sample concentration.

Induction of Diabetes
Healthy, adult male SD rats were used for the experiment.
Diabetes mellitus will be induced in overnight fasted rats by administration of single intraperitoneal (I.P.) injection of freshly prepared streptozotocin (STZ) with a dose of 60 mg/kg/mL [12].
Anti-hyperglycaemic activity of ethanolic extract of MR was studied using STZ-induced diabetic rats. The diabetic animals were showed decreases in body weight but the results were not significant ( Table   2). The diabetic rats showed significant increases in the levels of glucose throughout the study when compared with that of control   In the body weight analysis, the body weight changes between the groups was not remarkable. The increment of body weight was observed in the diabetic positive control groups treated with glibenclamide, 200 mg/kg and 400 mg/kg fruit extract whilst the body weight kept decreasing in the diabetic group due to the reason of increased muscle wasting as a consequence of increased protein catabolism. The observed body weight increment in the fruit extract treated groups might because of the antioxidant present, the phenolic groups that protected diabetic rats against stress, additionally helpful for recovery in body weight [13]. Conversely, the gradual increase of body weight in glibenclamide treated groups may be due to the growth of the skeletal size and epididymal fat [14].

Discussion
One the total phenolic content found was 6.420 mg GAE/g and 6.097 mg GAE/g for the concentrations of 50 and 200µg/ml respectively.
In this research work, the antioxidant potential of the MR extract was determined by using DPPH free radicals scavenging method and BHT was used as a standard. The DPPH is a stable freeradical which has a colour of purple, as the antioxidants react with the DPPH, the DPPH will be converted into non-radical DPPH-H form. Besides that, the colour of the DPPH will be decolorized from purple to light yellow colour. The degree of decolorization indicates the potency of the plant extracts in scavenging the free radicals. According to the results obtained, the IC50 was calculated for the BHT. The antibacterial activity of MR was tested by using the Escherichia coli, Staphylococcus aureus, Salmonella typhi, and Bacillus subtilis. The extract concentrations used in the agar well diffusion method were 0.1, 0.2, 0.4, 0.6, 0.8 and 1.0 mg/ml, other than that, 5mg/ml of penicillin and ciprofloxacin was playing the role as a standard in this study. In the present study, there was no zone of inhibition observed in all concentrations of ethanolic MR extract. The microorganism used were all in active condition, this can be proven as the ciprofloxacin as well as penicillin showed a clear zone of inhibition. In addition, the bacteria strains utilized were susceptible towards MR as stated [15][16][17][18][19][20], therefore it was concluded that the extracts have no antibacterial activity at the concentration of 0.1, 0.2, 0.4, 0.6, 0.8 and 1.0 mg/ml against gram negative. In the case of MIC test, the plant extract with the concentration of 0.1, 0.2, 0.4, 0.6, 0.8 and 1.0 mg/ml were utilized.

Conclusion
In conclusion, the phytochemical screening of ethanolic extracts of MR had determined the presence of alkaloids, flavonoids, carbohydrates and anthraquinones. The antibacterial activity of MR was determined by using the agar well diffusion and MIC assay.
The micro-organism used were Escherichia coli, Staphylococcus aureus, Salmonella typhi, and Bacillus subtilis. There was no antibacterial activity observed in the agar well diffusion with Escherichia coli and the MIC assay [21][22][23][24][25][26][27][28][29]. The antioxidant activity of the plant extract was assessed through the method of DPPH test while the total amount of phenolic compounds in the extract can be determined through the TPC test. It was suggested that MR can be a potential source as a natural significant antioxidant and less marked antimicrobial agent. The MR is a natural source that worth for further explore as it is inexpensive, natural, harmless and easy to obtain.

Interests of Conflict
The authors declare that there is no conflict of interests in the present publication.