INTRODUCTION:

Parkinson’s disease is the second most common neurodegenerative disease, primarily affecting people of ages over 55 years, although young adults and even children are also affected. Parkinson’s disease is characterized by the loss of 50–70% of dopaminergic neurons located in the substantia nigra. The neuropathological hallmark of Parkinson’s disease is the formation of eosinophilic Lewy bodies in surviving dopaminergic neurons. Current evidence suggests an involvement of both environmental and genetic factors in the progression of PD. The cardinal features of Parkinsonism are: bradykinesia, muscular rigidity, resting tremor, and an impairment of postural balance leading to disturbances of gait and falling.^{1, 2, 3, 4}

Melissa officinalis, a perennial bushy plant that reaches a height of 1m, belongs to the family, Lamiaceae.⁵ It is commonly referred to as balm, lemon balm and common balm. The major phytoconstituents present in Melissa officinalis are flavonoids: quercitrin, rhamncitrin, rhamnazin, Phenolic acids and tannins-rosmarinic acid (up to 4%), glycoside bound caffeic acid and chlorogenic acids, ferulic acid, hydroxycinnamic acid, protocatechuic acid and sesquiterpenes: β-caryophyllene oxide, geranyl acetate, linalool, eugenyl acetate, β-ocimene, copaene, and α-cubene.⁶ The plant has been widely used medicinally as carminative, digestive, diaphoretic, antioxidant, antiviral, antidepressant and stimulant activity⁷. The aim of the current study is to evaluate the neuroprotective activity of the extract of Melissa officinalis in MPTP induced Parkinson’s disease.

MATERIAL AND METHODS:

Plant:

Melissa officinalis was collected in the month of December from Dodabetta, The Nilgiris, Tamil Nadu, India. The collected plant material was authenticated by Dr. S. Rajan, Field Botanist, Survey of Medicinal Plants and Collection Unit, Central Council for Research in Homoeopathy, Dept. of AYUSH, The Nilgiris, Tamil Nadu.

Preparation of Extracts:

The Whole plant material of Melissa officinalis was dried under shade for 5 days and then was powdered coarsely. The powder (60g) was extracted successively with 500 mL of n-hexane, chloroform, ethyl acetate and ethanol. The obtained extracts were filtered and concentrated under reduced pressure using rotavapor to obtain the dried extracts.

Animals:

Healthy, adult Male albino mice (25-30 gm.) were obtained from the central animal house facility, J.S.S College of Pharmacy, Ootacamund, Tamil Nadu. The animals were housed in a well-ventilated room and the animals were exposed to 12 h day and 12 h night cycles, with a temperature between 20±3⁰C. All the experiments were performed after obtaining prior approval from IAEC.

Estimation of phenol content:

Total phenol content was determined colorimetrically using Folin-Ciocalteu reagent. 200μl of extract was mixed with 1.5 ml of Folin-Ciocalteu reagent (1:10 with distilled water), and allowed to stand at 22^oC±1^oC, for five minutes. A 1.5 ml sodium bicarbonate solution (8%) was added to the mixture. After 90 min at 22^oC±1^oC, absorbance was measured at 725 nm using a UV-Vis spectrophotometer. Total phenolic content was quantified by calibration curve obtained from measuring the absorbance of a known concentration of gallic acid standard (20-100μg/ml). The concentrations were expressed as mg of Gallic acid equivalents (GAE) per 100g of dry weight.⁸

In-vitro antioxidant study by DPPH method:

DPPH solution (0.2mg/ml) was freshly prepared using methanol as solvent; 0.5mL of this solution was mixed with 0.5mL of different dilutions (10, 50, 100, 150 and 200 𝜇g/mL) of the test sample. The volume of the solution was adjusted to 5mL with methanol. After incubation in the dark for 30 minutes at room temperature, the absorbance was measured at 517 nm. Butylated

Hydroxy toluene was used as standard. The antioxidant activity was evaluated by comparing the absorbance with the negative control (0.5mL of DPPH solution and 4.5mL of methanol).^{9, 10, 11,
12}

Percentage inhibition =100 – [A sample /A control X 100]

Acute toxicity study:

Acute oral toxicity was performed as per Organization for Economic cooperation for development (OECD) guideline 423 methods. Female non-pregnant rats were weighed and test substance was administered orally through gavage using specially designed rat oral needle. After the administration of test substance, food was withheld for 2 h but not water. Animals were observed individually after at least once during the first 30 minutes, periodically during the first 24hrs, with special attention given during the first 4 h, and daily thereafter, for a total of 14 days. Those animals which were found dead or in extreme distress, if any, are removed and humanely killed for animal welfare reasons.

Evaluation of Antiparkinsons activity of Ethanol extract in mice:

30 male Swiss albino mice, 8-12 weeks old, weighing 20-30 g were used for the study. They were divided into six groups of six animals each as given below

Group I: Normal (vehicle10ml/kg, p.o.)

Group II: Control

Group III: L-DOPA (13mg/kg p.o.)

Group IV: Ethanolic Extract (50mg/kg p.o.)

Group V: Ethanolic extract (100mg/kg p.o.)

Group VI: Ethanolic extract (200mg/kg p.o.)

All the animals from group I to VI were treated with respective assigned treatment for a period of 7 days. One week after treatment, on day 8 and 9 all the groups except group-I received MPTP (40 mg/kg, s.c) Treatment was continued for another 7 days post MPTP administration. The antiparkinson’s activity was evaluated by grip strength and locomotor activity. After these tests, the animals were sacrificed by ether anaesthesia. The brain was dissected and cortex, cerebellum, hippocampus and striata were separated for the estimation of lipid peroxidation, superoxide dismutase and catalase.

Evaluation of grip strength:

Rotarod was used to evaluate muscle grip strength by testing the ability of mice to remain on revolving rod. Rotarod apparatus was turned on and 20 rpm was set as an appropriate speed. Each mouse was given five trials before the actual reading was taken. The animals were placed individually one by one on the rotating rod. The ‘fall of time’ was noted when animal falls off from the rotating rod. The fall off time was then compared amongst the groups.¹³

Evaluation of locomotor activity:

The locomotor activity was measured using an actophotometer. The animals were placed individually in actophotometer for 10 minutes and basal readings were recorded. After induction of Parkinson’s disease with MPTP, the animals were again placed in actophotometer for 10 minutes to measure the locomotor activity. The change in activity before and after induction was calculated.¹⁴

Estimation of lipid peroxidation:

The tissue homogenate (0.2 mL), 0.2 mL of 8.1% sodium dodecyl sulphate (SDS), 1.5 mL of 20% acetic acid and 1.5 mL of 0.8% TBA were added. The volume of the mixture was made up to 4 mL with distilled water and then heated at 95°C on a water bath for 60 min. After incubation, the tubes were cooled to room temperature and final volume was made to 5 mL in each tube. 5 mL of butanol: pyridine (15:1) mixture was added and the contents were vortexed thoroughly for 2 min. After centrifugation at 3000 rpm for 10 min, the upper organic layer was taken and its optical density (OD) was measured at 532 nm against an appropriate blank without the sample. The levels of lipid peroxides are expressed as nmoles of thiobarbituric acid reactive substances (TBARS)/mg protein using an extinction coefficient of 1.56×105 ML cm-1.¹⁵

(OD X Volume of homogenate X 100 X10³)

nM of MDA/mg of tissue = ^{--------------------------------------------------------------------------------------------------------------------------------------------------------------------}

1.56 x10⁵ x volume of extract

Estimation of catalase:

Catalase in tissue was estimated as previously described by Beers and Sizer. 2.25 mL of potassium phosphate buffer (65 mM, pH 7.8) and 100 μL of the tissue homogenate/ sucrose (0.32 M) were incubated at 25^oC for 30 min. 0.65 mL of H2O2 (75 mM) was added to initiate the reaction. The change in absorption at 240 nm was measured for 2-3 min, and dy/dx for 1 min for each assay was calculated and the results expressed as CAT units / mg of tissue.

CAT (U) / 100 μL of Sample = [(dy/dx) x 0.003] / [38.3956 x10^-6]

The dy/dx (change in absorbance/min) was calculated for each assay divided by 38.3956 x 10^{– 6} (molar extinction coefficient of H2O2 at 240 nm) to obtain μM/l of H2O2 converted to H2O per min, multiplied by 0.003 to obtain micromoles.¹⁶

Estimation of Superoxide dismutase levels:

To an assay mixture containing 0.1 ml of supernatant, 1.2 ml of sodium pyrophosphate buffer (pH 8.3; 0.052 M), 0.1 ml of Phenazinemethosulphate (186 μm) and 0.3 ml of nitro blue tetrazolium (300 μM) 0.2 ml of NADH (750 μM) was added to start the reaction. After incubation at 30 ºC for 90s, the reaction was stopped by addition of 0.1 ml of glacial acetic acid. The reaction mixture was stirred vigorously with 4.0 ml of n-butanol. The intensity of Colour of the chromogen in butanol was measured spectrophotometrically at 560 nm and concentration of SOD was expressed as U/mg of protein.^{17, 18, 19, 20}

Estimation of complex-I activity:

The isolated tissue was homogenized with a Dounce tissue grinder in mitochondrial isolation buffer (70 mM sucrose, 210 mM Mannitol, 5 mMTrisHCl, 1 mM EDTA; pH 7.4) and suspensions were centrifuged at 800 g, 4C, for 10 min. The supernatant fluids were centrifuged at 13000 g, 4C, for 10 min, and the pellets were washed with mitochondrial isolation buffer and centrifuged at 13000 g, 4C, for 10 min to obtain the crude mitochondrial fraction. Brain mitochondria, isolated were lysed by freeze–thawing in hypotonic buffer (25 mM KH2PO4, 5 mM MgCl2, and pH 7.4). The reaction was initiated by the addition of 50 μg mitochondria to the assay buffer [hypotonic buffer containing 65 μM ubiquinone, 130 μM NADH, 2 μg/ml antimycin A and 2.5 mg/mL defatted bovine serum albumin (BSA)]. The oxidation of NADH by Complex I was monitored spectrophotometrically at 340 nm for 2 min at 30C. The activity was monitored for a further 2 min following the addition of rotenone (2 μg/ml). The difference between the rate of oxidation before and after the addition of rotenone was used to calculate complex-I activity.²¹

RESULTS:

Extraction:

The plant material was subjected to extraction with n-hexane, chloroform, ethyl acetate and ethanol. The maximum yield was obtained with ethanol followed by chloroform, n-hexane and ethyl acetate. The results are provided in Table I. The preliminary phytochemical evaluation of the extracts revealed the presence of steroids in n-hexane extract; alkaloids, flavonoids and saponins in chloroform extract; carbohydrates, flavonoids and terpenoids in ethyl acetate extract. Flavonoids, phenols and carbohydrates were found in ethanol extract.

Table I: Extraction of Melissa officinalis

S. No	Solvent	Percentage Yield (%W/W)
1	n-hexane	15%
2	Chloroform	27%
3	Ethyl acetate	6%
4	Ethanol	34%

Estimation of total phenol content:

The phenol content of ethanolic extractwas determined colorimetrically using Folin-Ciocalteu reagent. The total phenolic content of ethanolic extract of Melissa officinalis was found to be 15.1±3.45 g%. (gallicacid equivalent).

DPPH radical scavenging assay:

All the four extracts were assayed for their in-vitro antioxidant potential using DPPH radical scavenging assay. Among the four extracts, n-hexane and choloroform extract of Melissa officinalis did no exhibit any significant antioxidant effect. The ethyl acetate extract of Melissa officinalis showed weak antioxidant activity with an IC₅₀value of 635.46±28.45µg/ml. The maximum antioxidant activity (IC₅₀: 166.35±17.32) was exhibited by ethanolic extract of Melissa officinalis. The antioxidant activity shown by ethanolic extract is significantly higher than the standard, vitamin C (IC₅₀: 235.95±22.61). The results are summarized in Table II.

Table II: DPPH Assay of Melissa officinalis

S. No	Extract	IC50 (μg/ml)
1	n-hexane	-
2	Chloroform	-
3	Ethyl acetate	635.36± 28.45
4	Ethanol	166.35±17.32
5	Vitamin C (Standard)	235.95±22.61

Evaluation of antiparkinson’s activity:

Evaluation of grip strength:

The antiparkinson’s effect of ethanolic extract of Melissa officinalis was evaluated in parkinson’s disease induced animals using a rotarod. The effect of extract on retention time was determined at doses of 50mg/kg, 100mg/kg and 200mg/kg p.o. The ethanolic extract of Melissa officinalis did not show any significant effect at 50mg/kg p.o but showed a significant and dose dependent effect at 100mg/kg and 200mg/kg p.o. The results are summarized in Table: III

Table III: Effect of ethanolic extract of Melissa officinalison grip strength

S. No	Group	Retention Time
1	Normal	176.0 ±17.17
2	Control	80.17 ±14.34*
3	L-dopa	116.0 ± 23.71*
4	Test 50mg/kg	107.0 ±17.62^ns
5	Test 100mg/kg	111.0 ±20.07*
6	Test200mg/kg	132.2 ±34.63*

Values are expressed as mean ± SD. Statistical significance (p) was calculated by one way ANOVA followed by Tukeys multiple comparison test. Control group was compared with normal. Standard and test groups were compared with control. ^*P< 0.05 wereconsideredsignificant ; ns- not significant.

Evaluation of locomotor activity:

The antiparkinson’s effect of ethanolic extract of Melissa officinalis was evaluated in parkinson’s disease induced animals by measuring the locomotor activity using actophotometer. The effect of extract on locomotor activity was determined at doses of 50mg/kg, 100mg/kg and 200mg/kg p.o. The ethanolic extract of Melissa officinalis did not show any significant effect at 50mg/kg p.o but showed a significant and dose dependent effect at 100mg/kg and 200mg/kg p.o. The results are summarized in Table: IV

Table IV: Effect of Ethanolic extract of Melissa officinalis on the locomotor activity

S.no	Group	Locomotor activity (sec)
1	Normal	345.2±97.36
2	Control	202.8±30.66*
3	L-dopa	314.0±78.63*
4	Test 50mg/kg	305.8±68.39^ns
5	Test 100mg/kg	331.5±54.24*
6	Test200mg/kg	355.2±62.16*

In-vivo antioxidant study:

The effect of ethanolic extract of Melissa officinalis on lipid peroxidation and levels of SOD and catalase in cortex, cerebellum, hippocampus and striatum was estimated in MPTP induced Parkinson’s disease animals. The ethanolic extract of Melissa officinalis at 50mg/kg did not show any significant effect on lipid peroxidation or on the levels of SOD and catalase. The extract significantly prevented lipid peroxidation at 100mg/kg and 200mg/kg in hippocampus and striatum. MPTP significantly reduced the levels of SOD and catalase in control animals compared to normal. The test substance significantly restored the levels of SOD and catalase at 50mg/kg in cortex and striatum but it did not show any significant effect in cerebellum and hippocampus. The test substance significantly restored the levels of SOD and catalase at 100mg/kg and 200mg/kg in cortex, hippocampus and striatum but not in cerebellum.

Table V: Estimation of lipid peroxidation

S. No	Treatment groups	Cortex	Cerebellum	Hippocampus	Striatum
1	Normal	0.0100±0.00213	0.0138±0.00032	0.0091±0.0002	0.0063±0.00348
2	Control	0.01036±0.0035 ^ns	0.0101±0.0023^ns	0.09884±0.0006*	0.0103±0.0023*
3	Standard	0.0127±0.0056^ns	0.0113±0.0013^ns	0.01987±0.0004*	0.00905±0.006*
4	Test 50mg/kg	0.0122±0.00346^ns	0.0110±0.0013^ns	0.0200±0.0034^ns	0.00976±0.0026^ns
5	Test 100mg/kg	0.0124±0.0024^ns	0.0111±0.0067^ns	0.0165±0.0021*	0.00864±0.0002*
6	Test200mg/kg	0.0106±0.0034 ^ns	0.009493±0.0003^ns	0.0129±0.0034*	0.0077±0.000315*

Table VI: Estimation of superoxide dismutase

S.NO	Treatment groups	Cortex	Cerebellum	Hippocampus	Striatum
1	Normal	0.3826± 0.01294	0.3748±0.01471	0.7062±0.1096	0.7683±0.1036
2	Control	0.4510±0.004582*	0.2789±0.0001414^ns	0.3153±0.02475*	0.2366±0.05607*
3	Standard	0.3363± 0.01372*	0.2281±0.02157^ns	0.5211±0.02479*	0.4630±0.03516*
4	Test 50mg/kg	0.3309± 0.02242*	0.3004±0.006364^ns	0.4609±0.03656^ns	0.4492±0.0005233*
5	Test 100mg/kg	0.3083±0.008415*	0.2620±0.01478^ns	0.5412±0.004221*	0.5874±0.008337*
6	Test200mg/kg	0.2936± 0.01923*	0.2408±0.02362^ns	0.5412±0.06912**	0.6414±0.01082*

Table VII: Estimation of catalase

S. No	Treatment groups	Cortex	Cerebellum	Hippocampus	Striatum
1	Normal	1.478±0.07990	1.506±0.08888	0.7097±0.06689	4.710±0.06689
2	Control	0.7335±0.01421*	0.6798±0.07827*	0.1717±0.000650*	2.717±0.006505*
3	Standard	0.7629±0.01333*	0.7567±0.07814^ns	0.2862±0.001980*	3.686±0.1434*
4	Test 50mg/kg	0.7518±0.02906*	0.7079±0.07703^ns	0.2832±0.009267^ns	3.705±0.04971*
5	Test 100mg/kg	0.8079±0.006329*	0.4289±0.4007^ns	0.44070±0.01937*	4.247±0.1608*
6	Test 200mg/kg	0.9194±0.005614*	0.5108±0.1750^ns	0.5900±0.04171*	4.462±0.5052*

Mitochondrial complex I activity:

The mitochondrial complex I activity was estimated in all the groups after induction of Parkinson’s disease by administration of MPTP.The ethanolic extract of Melissa officinalis at doses of 50mg/kg, 100mg/kg and 200mg/kg significantly prevented MPTP induced inhibition of complex-I activity in brain.

Table VIII: Estimation of complex – I activity in mice brain

S. NO	Treatment Group	Complex I activity (nmol/min/mg protein)
1	Normal	91.12 ± 0.3272
2	Control group	64.40 ± 0.1697#
3	L-DOPA	65.17 ± 0.3070 *
4	Test 50mg/kg	77.14 ± 0.3646 *
5	Test 100mg/kg	78.50 ± 0.3939 *
6	Test200mg/kg	79.07 ± 0.3079 *

DISCUSSION:

The current studies on Parkinson’s disease states that oxidative stress is one of the major factors involved in the pathophysiology of this disease.Dopamine, metabolized to dopamine quinone, which itself acts as a source of oxidative stress causing damage to dopaminergic neurons. The dopamine quinone species affect the normal functions of GSH and protein cysteinyl residues which are very essential of cell survival. Dopamine quinone also alters the normal function of other proteins, like α-synuclein, parkin, DJ-1, and UCH-L1, that are linked to pathophysiology of parkinsonism. ^{22, 23}

Melissa officinalis has been reported extensively in the literature for its potent antioxidant activity.²⁴Melissa officinalis has also been reported for its potential use in neurodegenerative disease affecting the cerebral neurons. Recent evidence suggests that Melissa officinalis L. extract, which contains rosmarinic acid and the triterpenoids, oleanolic acid and ursolicacid, inhibits gamma-amino butyric acid transaminase (GABA-T) activity.²⁵

In the present study, n-hexane, chloroform, ethyl acetate and ethanolic extracts of Melissa officinalis was prepared and evaluated for their in vitro DPPH scavenging activity. Among the four extracts, ethanol extract showed highest free radical scavenging potential. It was, therefore, selected for in vivo studies. The ethanolic extract of Melissa officinalis was subjected to acute oral toxicity studies. The ethanolic extract of Melissa officinalis did not show any signs of toxicity or lethality even at 2000mg/kg. The antiparkinson’s activity of ethanolic extract was studied against MPTP induced changes in grip strength; locomotor activity; lipid peroxidation levels, superoxide dismutase levels and catalase levels in the cortex, the cerebellum, the hippocampus and the striatum.

MPTP is a contaminant in synthesis of heroin and induces Parkinson’s like syndrome in humans, nonhuman primates and in mice. Current models suggest that the neurotoxin crosses the blood-brain barrier following systemic administration and is oxidized to MPDP + by monoamine oxidase B. MPDP + is an unstable compound that undergoes further oxidation to form the active toxic compound MPP+. MPP + is actively transported into the dopaminergic neurons by the dopamine transporter. Within the dopaminergic neurons, MPP + sequesters inside the mitochondrial compartment because of the positive charge it carries and preferentially binds to and inhibits Complex I of the electron transport chain (ETC). Inhibition of complex I leads to lowering of ATP generation, increased production of reactive oxygen species (ROS), and eventual cell death.²⁶

The plant has been reported to have antioxidant properties and many flavonoids and other constituents such as hydroxycinnamic acid derivatives (rosmarinic and caffeic acids). The neuroprotection activity of ethanol extract may be therefore attributed to its antioxidant properties, which in turn may be due to the presence of flavonoid constituents in the ethanol extract.^{27, 28 29, 30} In conclusion the results of the present study clearly demonstrate the potential antiparkinson’s properties of the ethanol extract. The above extract, therefore, might be useful in the management of Parkinson’s disease.

The results of the present study clearly demonstrate the potential antiparkinson’s properties of the ethanol extract. The above activity may be attributed to its antioxidant properties which in turn may be due to the presence of flavonoids in the ethanol extract. Further, isolation and identification of specific phytoconstituents can help in identification of lead compounds that can be used in the treatment of Parkinson’s disease.

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Received on 28.11.2018 Modified on 10.01.2019

Research J. Pharm. and Tech. 2019; 12(5):2103-2108.

DOI: 10.5958/0974-360X.2019.00349.4