Murraya paniculata L. (Family: Rutaceae), grows commonly in the plain areas through Bangladesh⁹. It is known as cosmetic bark tree, is a native and common throughout much of India, Burma, and Malacca and dry areas of Ceylon and is often grown in Thailand, Cambodia, South Vietnam and East Africa¹⁰. They are armed shrub or small tree, leaves are 3 to 7 foliolate, and flowers are inflorescence, corymbose, fragrant. Traditionally most of the plant parts are used therapeutically in treatment of various diseases due to their emetic, antipyretic¹¹, carminative, anti-inflammatory¹², analgesic¹³ and antiulcer activities. Previous phytochemical studies showed two new coumarins from Murraya paniculata and showed mild butyl cholinesterase inhibitory activity^{14, 15}. Jou et al.¹⁶ identified the dioxin and dioxin-like polychlorbiphenyls in plant tissues and contaminated soils. Isoflavonoids in the Rutaceae family was found in Fortunella obovats, Murraya paniculata and in four Citrus species¹⁷. A new coumarin was also found in Murraya paniculata¹⁸. Structural features of a water soluble gum polysaccharide from Murraya paniculata in fruits was shown by Mondal et al.¹⁹. Kong et al.²⁰demonstrated the presence of a novel anti-implantation indole alkaloid. Chowdhury et al.²¹ reported that the oil of Murraya paniculata contain 58 compounds of which the major are caryophyllene oxide, β-caryophyllene, spathulenol, β-elemene, germacrene D and cyclooctene, 4-methylene-6-(1-propenylidene). Many studies have already been done with the isolated compounds and extractives of different active parts of this plant. However, pharmacological evaluation of bark extractives in terms of antioxidant and cytotoxic potential is largely unknown. Therefore, our present study was focused on the evaluation of antioxidant and cytotoxic potential of Murraya paniculata bark extractives to validate its traditional use.

MATERIAL AND METHODS:

Plant materials: Murraya paniculata (L.) bark was collected from the National Botanical Garden, Dhaka in March 2008 and was identified by Bangladesh National Herbarium, Mirpur, Dhaka, where a voucher specimen has been deposited. The bark of the plant was first sun dried and then ground into coarse powder.

Extraction of plant materials: The powdered plant material of bark (90 gm)of Murraya paniculata was extracted in a Soxhlet apparatus for 7 days with petroleum ether, 7 days with chloroform and 10 days with methanol. All the extracts were filtered through a cotton plug followed by Whatman filter paper number 1 and then concentrated by using a rotary vacuum evaporator to obtain petroleum ether (1.27 gm), chloroform (0.74 gm) and methanol (3.82 gm) extractives from the bark.

To get preliminary idea about the active constituents present in the plant bark extract different chemical tests were performed and showed the presence of alkaloid, flavonoids, steroids, gums and tannins²².

Antioxidant property

Qualitative assay: A suitably diluted stock solutions ( sample solutions) were spotted on pre-coated Silica gel TLC (Thin layer chromatography) plates and the plates were developed in solvent systems of different polarities (polar, medium polar and non-polar) to resolve polar and non-polar components of the extract and to choose the solvent system in which stock solutions run well. The plates were dried at room temperature and were sprayed with 0.02% DPPH (2, 2-diphenyl-1-picrylhydrazine) in ethanol. Bleaching of DPPH by the resolved bands was observed for 10 minutes and the color changes (yellow on purple background) were noted²³.

Quantitative assay: Free radical scavenging activity of the methanol extract was evaluated by determination of hydrogen peroxide scavenging activity, total antioxidant capacity, assay of nitric oxide scavenging activity and reducing power test. In all methods ascorbic acid is used as standard.

a. Scavenging of hydrogen peroxide: The ability of the methanolic extract of Murraya paniculata to scavenge hydrogen peroxide was determined according to the method of Ruch et al.²⁴. A solution of hydrogen peroxide (2 mmol/l) was prepared in phosphate buffer (pH 7.4). Hydrogen peroxide concentration was determined spectrophotometrically from absorption at 230 nm with molar absorbtivity 81 (mol/l)^-1 cm^-1 in a spectrophotometer (Hach, DR-4000U). Extracts (50–250mg/ml) in distilled water were added to a hydrogen peroxide solution (0.6 ml, 40 mM). Absorbance of hydrogen peroxide at 230 nm was determined after ten minutes against a blank solution containing in phosphate buffer without hydrogen peroxide. For each concentration, a separate blank sample was used for background subtraction. The percentage of scavenging of hydrogen peroxide of methanolic extract of Murraya paniculata and standard compounds was calculated using the following equation:

Percent scavenged [H₂O₂] = (A₀ − A₁)/ A₀ × 100

Where, A₀was the absorbance of the control, and A₁ was the absorbance in the presence of methanolic extract of Murraya paniculata and standards²⁵.

b. Determination of total antioxidant capacity: The antioxidant activity of the extract was evaluated by the phosphomolybdenum method according to the procedure of Prieto et al.²⁶. The assay is based on the reduction of Mo (VI) – Mo (V) by the extract and subsequent formation of a green phosphate / Mo (V) complex at acid pH. 0.3 ml extract was combined with 3 ml of reagent solution (0.6 M sulfuric acid, 28 mM sodium phosphate and 4 mM ammonium molybdate). The tubes containing the reaction solution were incubated at 95^οC for 90 min. Then the absorbance of the solution was measured at 695 nm using a spectrophotometer (Hach, DR-4000U) against blank after cooling to room temperature. Methanol (0.3 ml) in the place of extract is used as the blank. The antioxidant activity is expressed as the number of equivalents of ascorbic acid.

c. Assay of Nitric oxide scavenging activity: The procedure is based on the method, where sodium nitroprusside in aqueous solution at physiological pH spontaneously generates nitric oxide, which interacts with oxygen to produce nitrite ions that can be estimated using Greiss reagent. Scavengers of nitric oxide compete with oxygen leading to reduced production of nitrite ions. For the experiment, sodium nitroprusside (10 mM) in phosphate buffered saline was mixed with different concentrations of methanolic extract of Murraya paniculata dissolved in methanol and incubated at room temperature for 150 min. The same reaction mixture without the ethanol extract but the equivalent amount of methanol served as the control. After the incubation period, 0.5 ml of Griess reagent (1% sulfanilamide, 2% H₃PO₄ and 0.1% N-(1-naphthyl) ethylenediamine dihydrochloride was added. The absorbance of the chromophore formed was read at 546 nm²⁷.

d. Reducing power test: The reducing power of methanolic extract was determined according to the method of Oyaizu²⁸. Different amounts of methanolic extracts (100 - 1000 µg) in 1 ml of methanol were mixed with phosphate buffer (2.5 ml, 0.2 mol/l, pH 6.6) and potassium ferricyanide [K₃Fe(CN)₆] (2.5 ml, 1%). The mixture was incubated at 50^οC for 20 min. A portion (2.5 ml) of trichloroacetic acid (10%) was added to the mixture, which was then centrifuged (650 x g at room temperature) for 10 min. The upper layer of solution (2.5 ml) was mixed with distilled water (2.5 ml) and FeCl₃ (0.5 ml, 0.1%), and the absorbance was measured at 700 nm. Increased absorbance of the reaction mixture indicated increased reducing power.

Cytotoxicity study

Brine shrimp lethality bioassay^{29, 30} technique was applied for the determination of cytotoxic property of the crude petroleum ether, chloroform and methanol extractives of bark.

Preparation of positive control group: Vincristine sulphate was used as the positive control. Measured amount of the vincristine sulphate was dissolved in DMSO to get an initial concentration of 20 μg/ml from which serial dilutions were made using DMSO to get 10 μg/ml, 5 μg/ml, 2.5 μg/ml, 1.25 μg/ml, 0.625 μg/ml, 0.3125 μg/ml, 0.15625 μg/ml, 0.078125 μg/ml, 0.0390 μg/ml. Then then positive control solutions were added to the pre-marked vials containing ten living brine shrimp nauplii in 5 ml simulated sea water to get the positive control groups.

Preparation of negative control group: 100 μl of DMSO was added to each of three pre-marked glass vials containing 5 ml of simulated sea water and 10 shrimp nauplii to use as control groups. If the brine shrimps in these vials show a rapid mortality rate, then the test is considered as invalid as the nauplii died due to some reason other than the cytotoxicity of the compounds.

Counting of nauplii: After 24 h, the vials were inspected using a magnifying glass and the numbers of survived nauplii in each vial were counted. From this data, the percent (%) of lethality of the brine shrimp nauplii was calculated for each concentration.

RESULTS:

Antioxidant property:

Qualitative assay: The color changes (yellow on purple background) on the TLC plates were observed due to the bleaching of DPPH by the resolved bands.

Quantitative assay:

a. Scavenging of hydrogen peroxide: Scavenging of H₂O₂ by extracts may be attributed to their phenolic constituents, which can donate electrons to H₂O₂, thus neutralizing it to water. The methanolic extract of Murraya paniculata was capable of scavenging hydrogen peroxide in a concentration-dependent manner (Figure 1).

Figure 1: H₂O₂ scavenging activity of methanolic extract of Murraya paniculata Vs Ascorbic acid

b. Total antioxidant capacity: Total antioxidant capacity exerted by the extract was concentration dependent. It was observed that the extract was likely to have the capacity of reduction of Mo (VI) to Mo (V) by the antioxidant principle and the formation of a green phosphate / Mo (V) complex with a maximal absorption at 695 nm. The antioxidant activity is expressed as the number of equivalents of ascorbic acid (Table 1).

Table 1: Total antioxidant capacity of methanolic extract of Murraya paniculata

Materials

Concentration (μg/mL)

Equivalent to ascorbic acid

Methanol extract of Murraya paniculata

125

250

500

0.081±0.17

0.172±0.13

0.287±0.08

0.731±0.15

1.532±0.09

3.211±0.11

c. Nitric oxide scavenging activity: From Figure 2, it is observed that the extract is likely to have concentration dependent nitric oxide scavenging activity. The bark may have the property to counteract the effect of NO formation and in turn may be of considerable interest in preventing the ill effects of excessive NO generation in the human body. Further, the scavenging activity may also help to arrest the chain of reactions initiated by excess generation of NO that are detrimental to the human health. Nitric oxide is also implicated for inflammation, cancer and other pathological conditions³¹.

Figure 2: Nitric oxide scavenging activity of methanolic extract of Murraya paniculata

d. Reducing power activity: Reduction ability of the extract has been investigated from the Fe⁺⁺⁺ - Fe⁺⁺ transformation using the method followed by Oyaizu²⁸. Earlier authors^{32, 33}have observed a direct correlation between antioxidant activity and reducing power of certain plant extracts. The reducing properties are generally associated with the presence of reductones³² which have been shown to exert antioxidant action by breaking the free radical chain by donating a hydrogen atom³⁴. Reductones are also reported to react with certain precursors of peroxide, thus preventing peroxide formation. Figure 3 shows the reduction ability of Murraya paniculata.

Figure 3: The reducing power of methanolic extract of Murraya paniculata

Cytotoxic effect: Following the procedure of Meyer et al.³⁵ the lethality of the crude extract to brine shrimp was determined on Artemia salina. Table 2 shows the results of the brine shrimp lethality testing after 24 hours of exposure to the petroleum ether, chloroform and methanolic extract and the positive control, vincristine sulphate. The LC₅₀ obtained from the best-fit line slope was found to be 0.84±0.19 µg/ml, 1.17±0.03 µg/ml, 1.31±0.16 µg/ml respectively in comparison with Vincristine Sulphate (LC₅₀: 0.33±0.12 µg/ml).

Table 2: LC₅₀ data of the test sample of Murraya paniculata in brine shrimp lethality bioassay

Plant part	Crude extract	LC₅₀ ( µg/ml)
Bark	Petroleum ether	0.84±0.19
	Chloroform	1.17±0.03
	Methanol	1.31±0.16
Standard	Vincristine Sulphate	0.33±0.12

DISCUSSION:

Cancer is perhaps the most progressive and devastating disease posing a threat of mortality to the entire world despite significant advances in medical technology for its diagnosis and treatment. All cells are exposed to oxidative stress, and thus oxidation, and free radicals may be important in carcinogenesis at multiple tumor sites⁵. Antioxidants with free radical scavenging activities have great relevance in the prevention and therapeutics of diseases in which oxidants or free radicals are implicated⁷. Phytochemicals are one of the major sources of antioxidants, which are potentially capable to scavenge free radicals to decrease the risk of many chronic diseases such as cancer and cardiovascular disorders³⁶. Therefore, over the last few decades herbal antioxidants have been gaining prime importance in the antiradical drug discovery due to their lesser side effects as reviewed extensively by many authors³⁷. Further, cytotoxic effect of natural bioactive compounds specify a wide range of pharmacological activities such as antimicrobial, antiviral, pesticidal and anti-tumor etc. of the compounds³⁵ and thus the cytotoxic compounds isolated from natural resources are good candidates for anticancer drugs³⁸. Since, Murraya paniculata bark extractives showed significant free radical scavenging tendencies in all the experimental methods and all extractives showed potent cytotoxic effect; it can be a good candidate in future anticancer drug development.

CONCLUSION:

Our present study reveals the antioxidant and cytotoxic potential of Murraya paniculata (L.) bark, which can be a scientific avenue to proceed further exhaustively to explore its active constituents responsible for antioxidant and cytotoxic potential. We believe, further detailed advanced studies may explore novel leads for anticancer drug.

ACKNOWLEDGMENTS:

Authors wish to thank the authority of Stamford University Bangladesh for extending their cordial supports to perform these investigations.

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Received on 16.12.2011

Modified on 27.12.2011

Accepted on 01.01.2012

Research Journal of Pharmacognosy and Phytochemistry. 4(1): Jan. - Feb. 2012, 18-22