Assessment of phytotoxic, genotoxic and enzyme inhibition potential of Sterculia diversifolia

Abstract

OBJECTIVE:

To evaluate Sterculia diversifolia stem bark and leaves for phytotoxic, genotoxic and enzymes inhibition potential.

METHODS:

Phytotoxic activity of both stem bark and leaves were screened using Lemna minor. The genotoxic activity of Sterculia diversifolia stem bark and leaves extracts were tested using comet assay protocol while enzyme inhibition activity of crude extract and various fractions of both stem bark and leaves were evaluated using acetyl cholinesterase, lipoxygenase, β-glu-curonidase, urease, xanthine oxidase and carbonic anhydrase.

RESULTS:

Phytotoxic activity showed significant results in dose dependant manner in both stem bark (ethyl acetate and n-butanol) and leaves (ethyl acetate, n-butanol and n-hexane) fractions. In genotoxic activity, dichloromethane fraction showed significant activity followed by ethyl acetate fraction. Acetyl cholinesterease inhibitory activity showed significant results in both stem bark and leaves fractions, while significant lipoxygenase inhibition was shown by ethyl acetate, dichloromethane, crude extract and n-hexane fractions of both stem bark and leaves. β-glucuronidase, urease and carbonic anhydrase inhibitory activity showed highly significant results in ethyl acetate fraction of both stem bark and leaves, while xanthine oxidase inhibition was shown by dichloromethane fraction of stem bark and leaves extracts.

CONCLUSION:

This study emphasizes the important phytotoxic, genotoxic and enzyme inhibition effects of Sterculia diversifolia stem bark and leaves. Hence, it is clear that Sterculia diversifolia stem bark and leaves possess phytotoxic, genotoxic and enzyme inhibitory agents.

1. INTRODUCTION

Nature is a serving man for treating diseases since paleolthic ages. Medicinal plants are rich sources of therapeutic moieties and are used for curing diseases.¹ A number of plants and their extracts have been used in traditional medicines.^2,3 Drugs used in current practices have been derived from natural sources either directly or indirectly.⁴ Excellent therapeutic moieties could be discovered by detailed extensive research on medicinal plants. These moieties should be effective, less toxic and highly potent in resistant pathological conditions.⁵

Sterculiaceae family comprised of 60 genera and 1500 species belonging to tropical as well as sub-tropical region.⁶ This family is a source of bioactive constituents of various chemical classes e.g. polyphenols, flavonoids, glycosides, alkaloids, steroids, terpenoids, triterpenes, saponins, sterols, apigenins, tannins, essential oils, carbohydrates and proteins.^7,8 Medicinally, Sterculia diversifolia bears antimicrobial, immune-omodulatory, cytotoxic, anti-glycation, larvicidal, anthelmintic, leishmanicidal, insecticidal, antioxidant, anticonvulsant, CNS depressant, analgesic, antipyretic, anti-infla-mmatory, laxative, anti-diarrheal, hepat-oprotective and diuretic activity.^{9,⇓,⇓,⇓-13} Various secondary metabolites have been reported from Sterculia diversifolia e.g. Stercularin, luteolin, isoquercitrin, ursolic acid, gossypetin, taxifolin, methyl 4-hydroxycinnamate and β-sitosterol-D-glucoside.^9,12,14 This plant also bears metal contents and fatty acid constituents as reported in the literature.^15,16 The present study was designed to explore phytotoxic, genotoxic and enzyme inhibitory potential of medicinally important plant, Sterculia diversifolia.

2. MATERIALS AND METHODS

2.1. Plant material

Stem bark and leaves were collected in September, 2014 from Pakistan Forest Institute botanical garden (34 00'50.6"N, 71 29'03.0"E), University of Peshawar, Peshawar, Pakistan. Ghulam Jelani (taxonomist) at the Department of Botany, University of Peshawar identified the plant and deposited a specimen in their herbarium under the reference No. Bot. 20098 (PUP).

2.2. Extraction and fractionation

Sterculia diversifolia stem bark and leaves (17 and 13 kg respectively) were dried under shade at room temperature. After grinding to powder, macerated for two weeks with solvent (hydromethanolic: 10%-90%). Filtration was conducted with filter paper (Whatman-I) after maceration. Rotary evaporator was used for the concentration of obtained crude extracts under reduced pressure at 40 ℃.¹⁷ The obtained crude extracts of both parts were 950 g and 1.2 kg respectively. Methanolic extract of Sterculia diversifolia (MESD) stem bark and leaves were then soaked overnight after mixing with 2.5 L distilled water. Various organic solvents were used in fractionation i.e. n-hexane (3 × 5 L), DCM (3 × 5 L), ethyl acetate (3 × 5 L) and n-butanol (3 × 5 L). The remaining residue was considered as aqueous fraction.

2.3. Phytotoxic activity

The MESD (stem bark and leaves) and its fractions were evaluated for phytotoxic potential using standard protocol.¹⁸ The MESD and its fractions were added with sterilized E-medium at various concentrations (10, 100 and 1000 µg/mL) in methanol. Inoculation of sterilized conical flask with desired concentrations fraction prepared from the stock solution and evaporated overnight. The inoculation of each flask was conducted with sterilized E-medium (20 mL). Ten Lemna minor were selected which were placed on media, each containing three frond rosette. Methanol and Parquet were inoculated in other flasks serving as negative and positive control. Treatment was conducted in triplicate and incubated the flasks for a week at 30 ℃ in growth cabinet (Fisons Fi-Totron 600H), intensity (9000 lux), relative humidity (56 + 10 rh) and at a day length of 12 h. Lemna minor growth in fraction containing flask was calculated by counting fronds number per dose. The determination of growth inhibition was achieved with reference to negative control. Percent (%) growth inhibition was determined by the given formula;

% inhibition = 100 － (Number of fronds in test sample) / (Number of fronds in control) × 100

2.4. Genotoxic activity

Genotoxic potential of crude extracts and various subsequent fractions of both stem bark and leaves extract (20, 100, 200 mg/L) was determined by comet assay.¹⁹ Clean glass slides (n = 36) were taken and agarose gel (0.75%) of low melting point was poured (80 µL) on them. Then agarose gel was allowed for solidification for about 45 min. Lymphocytes were poured (100 µL) into 96 well plate which was previously separated from sheep blood and then incubated for 2 h with selected concentration at 37 ℃. Each well (100 µL) containing suspension, which was pipetted out and poured on prepared agarose gel slides. Then on each slide agarose gel (0.5%) of low melting point was poured (80 µL) and allowed for solidification. Lyses solution (500 mL) was kept under refrigeration for 12 h in which slides were dipped. Lyses solution was removed on the 2nd day and placed the slides for 25-45 min in alkaline buffer (500 mL). Slides were kept in electrophoresis chamber having buffer [boric acid MP biomedicals, LLC (5.5 g), Tris base (10.8 g) and disodium EDTA (0.93 g) were dissolved in double distilled water (700 mL) and made the final volume upto 1000 mL] and ran for 45 min at 25 v. From electrophoresis chamber slides were removed and washed two times with neutralizing buffer [24.25 g Tris base was mixed with double distilled water (400 mL) and made the final volume upto 500 mL] and then stained with ethidium bromide solution (50-80 µL). Under fluorescent microscope slides were observed at 100 × magnification with 590 nm barrier filter and 560 nm excitation filter. Doxorubicin hydrochloride and lymphocytes were used as positive control and negative control respectively. The classes of comets were; Class 0 (without tail nucleus), Class 1 (tail less than nucleus diameter), Class 2 (tail one to two times the nucleus diameter) and Class 3 (tail > 2 time the nucleus diameter). The experiments were conducted in triplicate and per treatment, 300 cells were analyzed. The trypan blue exclusion method determination of cell viability was conducted, where only treatments with > 80% viability were considered. The number of cells multiplication in each class by the damage class gave DNA damage score for each sample. DNA damage score was calculated according to the formula:

Total DNA damage score = (0 × n⁰) + (1 × n¹) + (2 × n²) + (3 × n³)

where n = each class cells number. Thus, the total score could range from 0 to 300.

2.5. Enzyme inhibition activity

2.5.1. Acetyl cholinesterase inhibitory assay

Modified spectrophotometric method was used for determining the acetyl cholinesterase (AChE) inhibitory activity.²⁰ Electric-eel AChE (type VI-S, Sigma, St. Louis, MO, USA) was used as source of cholinesterases and the substrate for AChE was acetylthiocholine iodide to perform reaction. Cholinesterase activity was measured and monitored using 5, 5-Dithiobis (2-nitrobenzoic acid) (DTNB, Sigma, St. Louis, MO, USA). Both the tested sample and the standard were dissolved in ethanol. Reaction mixture contained 10 μL of DTNB, 150 μL of sodium phosphate buffer (100 mM, pH 8.0), 20 μL of acetylcholinesterase and 10 μL of sample solution were mixed and incubated at 25 ℃ for 15 min. The reaction was initiated by dissolving 10 mL of acetylthiocholine. A yellow 5-thio-2-nitrobenzoate anion was formed by the enzymatic hydrolysis of acetythiocholine which results by the reaction of DTNB and thiocholine at 412 nm, while kept for 15 min. These reactions took place in 96-well micro-plate in triplicate. The percentage inhibition of AChE was determined by using the formula given below,

% inhibition = (E − S) / E × 100

Where, E and S is the activity of the enzyme without test sample and with test sample respectively. The IC₅₀ values were measured by EZ-Fit Enzyme Kinetics program and expressed in mean ± standard error of mean (SEM) (μM).

2.5.2. Lipoxygenase inhibition assay

The modified spectrometric method of Babatunde et al ²¹ was used for lipoxygenase assay. The linoleic acid and Lipoxygenase (EC 1.13.11.12) type I-B i.e., Soybean were taken from Sigma (St. Louis, MO) in addition to all other chemicals. Reaction mixture consists of 160 μL of sodium phosphate buffer (0.1 mM; pH 7.0), sample solution of 10 mL and lipoxygenase solution of 20 μL. At 258 ℃, the mixture was incubated for 5 min. Reaction was intiated by linoleic acid substrate solution (10 μL) addition. Absorption changes with the formation of (9Z, 11E)-13S)-13-hydroperoxyoctadeca-9, 11-dienoate after 10 min. In 50% ethanol, test sample (i.e. extract, fractions) and the standard were dissolved and then analysed in triplicate. The standard used for lipoxygenase inhibition was Baicalein.

2.5.3. β-Glucuronidase inhibition assay

MESD (stem bark and leaves) and its fractions were screened for β-Glucuronidase enzyme inhibition using spectrophotometric method of Medina-Perez with minor modification.²² A substrate p-nitrophenyl-β-D-glu-curonide (250 μL) was used for this assay during which p-nitrophenol formed from substrate, was confirmed by absorbance at 405 nm and added to 96-well micro plate. The reaction mixture containing test sample (10 μL), enzyme solution (10 μL) and acetate buffer (0.1 m, 185 μL). Reagents were mixed with the help of DMSO. The incubation period for the reaction mixture was 30 min at 37 ℃. p-nitrophenyl-β-D-glucuronide (0.4 mM, 50 μL) was added to the reaction mixture and then change in absorbance was measured for 30 min continuously at 405 nm with Spectra Max spectro-photometer. Following formula was used for the determination of percent inhibition.

% inhibition = 100 - (Optical Density test sample/ Optical Density control) x 100

The IC50 values were determined using the EZ-Fit enzyme kinetic program

2.5.4. Urease inhibition assay

MESD (stem bark and leaves) and its fractions were screened using the procedure of Weatherburn for urease inhibitory assay.²² The reaction mixture [enzyme solution (Jack bean 25 μL) and buffer solution (55 μL) containing urea (100 mM)] along with each test sample (5 μL, 1 mM) was incubated for 15 min at 30℃ in 96-well plate. Ammonia produced during reaction was measured using indophenol’s procedure to determine urea activity. Alkali reagent (70 μL) and phenol reagent (45 μL) were added to each well. Alkali reagent was prepared from NaOH (0.5% w/v) and active chloride NaOCl (0.1%), while phenol reagent was prepared from phenol (1% w/v) and sodium nitroprusside (0.005 % w/v). Absorbance was measured at 630 nm after 50 min with the help of micro plate reader. Change (per min) in absorbance was recorded with Soft-Max Pro software. EDTA (1.0 mM), LiCl₂ (0.01 M) and K₂HPO_4.3H₂O (0.01 M) was added to maintain the pH at 8.2. Thiourea was used as control while whole experiment was performed in triplicate. Following formula was used for the determination of percent inhibition.

Percent inhibition = 100 - (Optical Density test well / Optical Density control) ×100

2.5.5. Xanthine oxidase (XO) inhibition assay

This assay was used for MESD (stem bark and leaves) and its fractions screening using spectrophotometric method reported by Samaha et al. with minor modification.²³ Methanol-phosphate buffer (1%) was used for the dissolution of sample (i.e. extracts, fractions) to make up 100 μg/mL final concentration. Reaction mixture was prepared containing test samples (100 μL), enzyme solution (100 μL) and phosphate buffer (300 μL, 0.2M, pH 9), then it is incubated (2 min) at room temperature. The reaction was initiated by adding xanthine oxidase solution (500 μL) prepared in phosphate buffer (0.15 mM). Spectrophotometer was used for the determination of absorbance (295 nm) at room temperature for 2 min. In this assay allopurinol (100 μg/mL) was used as a positive control. Following formula was used for the determination of percent inhibition.

% inhibition = (Change in Abs. control - Change in Abs. test sample) × 100) / Change in Abs. control

2.5.6. Carbonic anhydrase inhibition assay

Carbonic anhydrase assay was performed for MESD (stem bark and leaves) and its fractions screening. Production of yellow colour 4-nitrophenol and colourless 4-nitrophenylacetate upon hydrolysis was measured in this assay.²⁴ Reaction was carried out in buffer solution (HEPES-tris, 20 mM, pH 7.2-7.9) at 25-28 ℃. Buffer (140 μL) and purified bovine erythrocyte (20 μL) was added in each sample tube solution. 4-nitrophenylacetate substrate (20 μL, 0.6-0.8 mM) and extracts were dissolved in DMSO (10%) and then diluted in ethanol. The reaction was carried out using 96- well micro plate. Product formation rate was monitored for 30 min keeping 1 min interval by using microplate readers. Percent inhibition was determined by following formula;

% inhibition = 100 - (Optical Density test sample/ Optical Density control) × 100

3. RESULTS

3.1. Phytotoxic activity

Crude MESD and various fractions of stem bark and leaves were evaluated for phytotoxic activity at 1000, 100 and 10 μg/mL concentrations. The paraquat was used as standard phytotoxic drug (0.015 µg/mL). The results of test samples were concentration dependent. In crude MESD and subsequent fractions of stem bark, ethylacetate and n-butanol fractions showed maximum percent growth inhibition at 1000 μg/mL concentration i.e. 100% and 80% respectively. While in case of crude MESD and its fractions of leaves, EtOAc, n-butanol and n-hexane fractions showed maximum percent growth inhibition at 1000 μg/mL concentration i.e. 85%, 85% and 80% respectively as presented in Table 1.

Table 1.

Phytotoxic activity of crude extracts (stem bark and leaves) and its fractions

Test Sample	No. of fronds (3 fronds per plant)	Sample conc. (µg/mL)	Stem bark				Leaves
			No. of died fronds	% Growth Inhibition		No. of died fronds		% Growth Inhibition
MESD	20 20 20	10 100 1000	01 02 09	05 10 45		05 07 14		25 35 70
n-Hexane	20 20 20	10 100 1000	03 04 07	15 20 35		03 05 16		15 25 80
DCM	20 20 20	10 100 1000	04 05 06	20 25 30		02 04 12		10 20 60
Ethylacetate	20 20 20	10 100 1000	03 04 20	15 20 100		02 03 17		10 15 85
n-Butanol	20 20 20	10 100 1000	01 04 16	5 20 80		04 07 17		20 35 85
Aqueous	20 20 20	10 100 1000	02 03 04	10 15 20		03 06 14		15 30 70

Notes: MESD: methanolic extract of Sterculia diversifolia; DCM: dichloromethane.

3.2. Genotoxicity activity

Genotoxic activity of MESD (stem bark and leaves) and its fractions were evaluated. Table 2 showed level of damage as well as damage index (DI) by the tested samples in three different concentrations used. The level of damage was significant for DCM and ethyl acetate fractions, while DCM and ethyl acetate fractions also showed significant damage index at all concentrations especially at higher concentrations. The data of genotoxicity effects were also comparable to those of the doxorubicin hydrochloride (positive control) used in this study.

Table 2.

Genotoxicity activity of crude MESD stem bark, leaves and its fractions

Test Sample	Conc (mg/L)	Stem bark						Leaves
		Level of Damage					DI	Level of Damage					DI
		0	1	2		3		0		1	2	3
MESD	20	68.46±0.62	21.59±0.89	7.23±1.83		2.72±0.81	44.21		64.68±1.23	29.27±0.55	5.30±0.38	1.75±0.19	45.12
	100	62.00±0.50	19.91±1.80	11.17±0.37		6.92±0.11	57.01		61.70±0.42	25.61±1.80	9.87±0.97	2.82±1.81	53.81
	200	60.29±1.73	30.55±0.22	6.90±0.01		2.26±1.48	45.13		59.92±0.27	31.65±0.79	7.11±0.61	1.32±0.22	49.83
n-hexane	20	77.13±0.97	21.59±1.29	1.30±0.12		0.98±1.18	27.13		79.22±0.54	18.71±0.58	1.10±0.10	0.97±0.18	23.82
	100	72.40±1.30	20.13±0.92	5.15±0.74		1.73±0.45	35.62		75.39±0.61	16.87±0.67	6.12±0.26	0.62±0.20	30.97
	200	74.25±1.64	18.61±0.88	6.14±0.96		1.00±0.92	33.89		71.64±1.98	22.43±0.82	4.88±0.42	1.05±0.23	35.34
DCM	20	60.61±0.79	33.67±0.78	4.60±0.54		1.12±1.14	46.23		50.78±1.77	40.88±1.09	7.16±0.94	1.18±0.12	58.74
	100	54.30±0.05	38.12±1.11	5.75±1.19		1.59±0.86	54.39		44.20±0.75	37.82±1.39	15.22±1.00	2.76±1.01	76.54
	200	52.23±0.33	25.45±0.22	16.77±0.12		5.55±1.76	75.64		45.29±0.34	38.12±0.95	14.12±0.93	2.78±0.13	74.70
EtOAc	20	59.32±0.22	29.56±1.79	09.31±0.34		1.87±0.54	53.79		58.90±1.29	30.07±0.57	8.12±0.66	2.91±0.46	55.04
	100	52.10±0.50	40.22±0.89	5.20±0.87		2.48±0.67	58.06		50.90±0.88	43.76±0.23	4.26±0.22	1.08±0.33	55.52
	200	53.65±0.88	32.72±0.42	11.87±0.12		1.24±1.65	60.18		47.05±0.12	42.12±0.40	9.00±1.00	1.83±0.64	65.61
n-butanol	20	86.36±0.34	10.22±0.80	1.96±1.77		1.50±0.28	18.64		83.02±0.68	13.72±0.58	2.05±0.43	1.21±0.20	21.45
	100	79.19±1.31	16.11±1.83	2.99±0.09		1.71±0.78	27.22		80.99±1.69	14.51±0.84	3.29±0.28	1.21±0.16	24.72
	200	77.33±0.66	14.12±0.73	6.92±0.01		1.63±0.86	32.85		77.98±1.93	17.12±0.56	3.22±0.67	1.68±0.77	28.60
Aqueous	20	88.66±1.55	7.32±0.23	3.02±0.51		1.00±0.58	16.36		84.01±1.89	10.67±1.80	4.06±0.22	1.10±0.47	22.09
	100	78.29±0.67	15.31±0.99	5.34±1.37		1.06±0.22	29.17		77.41±1.34	18.58±1.36	3.12±0.47	0.81±0.12	27.25
	200	79.54±0.52	11.79±1.44	5.88±0.01		3.66±0.14	34.53		72.32±1.90	21.32±1.06	4.19±0.60	2.17±1.00	36.21
-ive control	0	84.05±1.20	13.67±0.43	2.28±0.63		0.00±0.00	18.23		84.05±1.20	13.67±0.43	2.28±0.63	0.00±0.00	18.23
+ive control	4mM	11.00±2.03	48.21±0.99	25.22±1.32		15.59±2.63	145.42		11.0±2.03	48.21±0.99	25.22±1.32	15.59±2.63	145.42

Notes: MESD: methanolic extract of Sterculia diversifolia; DCM: dichloromethane; DI: damage index; EtOAc: ethyl acetate.

3.3. Enzyme Inhibition activity

3.3.1. Acetylcholinesterase inhibitory assay

The crude MESD (stem bark and leaves) and its fractions were screened for Acetylcholinesterase inhibitory activity. The maximum inhibitory activity of stem bark extract and fractions were observed for n-hexane (98.25%) with IC₅₀ value of (1.32 ± 0.20) µg/mL, followed by EtOAc (93.50%) with IC₅₀ value of (22.08 ± 0.14) µg/mL, DCM (90.50%) with IC₅₀ value of (40.40 ± 0.22) µg/mL, crude MESD (86.60%) with IC₅₀ value of (55.22 ± 0.10) µg/mL, n-butanol (82.10%) with IC₅₀ value of (60.34 ± 0.20) µg/mL and aqueous fraction (80.30%) with IC₅₀ value of (62.28 ± 0.34) µg/mL) respectively as shown in Table 3. The maximum inhibitory activity of leaves extract and fractions were observed for n-hexane (96.84%) with IC₅₀ value of (3.69 ± 0.58) µg/mL, followed by EtOAc (94.92%) with IC₅₀ value of (18.78 ± 0.20) µg/mL, DCM (91.24%) with IC₅₀ value of (38.06 ± 0.14) µg/mL, crude MESD (84.06%) with IC₅₀ value of (58.28 ± 0.18) µg/mL, aqueous fraction (81.22%) with IC₅₀ value of (60.55 ± 0.30) µg/mL and n-butanol (78.86%) with IC₅₀ value of (70.68 ± 0.68) µg/mL respectively as shown in Table 3. The Acetylcholinesterase inhibitory activity of standard drugs (Galathamine) was 0.5 µM respectively.

Table 3.

Enzymes Inhibitory potential of crude MESD and its fractions

Sample		Acetylcholinesterase				Lipoxygenase		β- glucuronidase			Urease			Xanthine oxidase						Carbonic anhydrase
		Inhibition (%)	IC50± SEM (µM)			Inhibition (%)	IC50± SEM (µM)		Inhibition (%)	IC50± SEM (µM)		Inhibition (%)	IC50±SEM (µM)			Inhibition (%)	IC50± SEM (µM)				Inhibition (%)	IC50±SEM (µM)
Stem bark	MESD	86.60	55.224± 0.103		88.50		54.624±0.402	11.50		135.524±0.202	49.80		502.486± 0.902		58.48			428.244±1.805	60.60			250.262±0.408
	n-Hexane	98.25	1.322 ± 0.204		77.50		70.278±0.330	32.60		116.343±0.165	33.80		Not active		30.40			Not active	26.50			Not active
	DCM	90.50	40.405± 0.224		92.10		48.435±0.176	40.65		106.550±0.127	38.52		Not active		84.62			108.424±0.888	46.38			495.825±0.167
	EtoAc	93.50	22.083± 0.148		92.75		37.244±0.303	70.60		56.148± 0.263 132.624±0.286	83.67		116.426 ±1.296		76.34			194.282±1.847	78.28			182.286±0.103
	n-butanol	82.10	60.348± 0.204		61.75		98.303±0.105	12.82			55.33		458.243± 2.100		48.86			514.520±1.059	35.85			538.70±0.200
	Aqueous	80.32	62.285± 0.345		55.23		106.623±0.120	16.12		124.083±0.405	42.62		526.602± 3.425		36.31			Not active	15.34			Not active
Leaves	MESD	84.06	58.284± 0.188		86.98		60.249±0.329	17.20		125.463±0.484	47.85		508.153± 1.745		59.64			424.322±1.266	68.24			206.024±1.282
	n-Hexane	96.84	3.690± 0.584		71.44		80.840±0.675	38.38		106.543±0.330	30.58		Not active		32.58			Not active	36.78			Not active
	DCM	91.24	38.063± 0.148		94.88		40.080±0.708	43.34		101.442±0.666	31.66		Not active		89.20			96.420±1.808	59.32			406.702±2.526
	EtoAc	94.92	18.785± 0.202		94.55		42.804±0.927	74.68		50.302± 0.643	82.33		128.502± 1.089		78.55			190.325±1.005	83.48			154.262±0.980
	n-butanol	78.86	70.687± 0.684		43.85		119.342±0.963	13.98		130.220±0.196	51.92		480.344± 1.564		52.60			480.228±1.085	49.04			500.643±0.902
	Aqueous	81.22	60.554± 0.306		39.90		124.287±0.720	18.02		122.502±0.253	32.70		Not active		37.58			Not active	35.12			Not active
Galathamine		99.80	0.500± 0.073		ND		ND	ND		ND	ND		ND		ND			ND	ND			ND
Baicalein		ND	ND		88.2		22.002± 0.052	ND		ND	ND		ND		ND			ND	ND			ND
D-Saccharic acid 1,4 lactone		ND	ND		ND		ND	89.45		48.789± 2.163	ND		ND		ND			ND	ND			ND
Thiourea		ND	ND		ND		ND	ND		ND	98.2		21.046± 0.0113		ND			ND	ND			ND
Allopurinol		ND	ND		ND		ND	ND		ND	ND		ND		98.6			2.002±0.0182	ND			ND
Acetazolamide		ND	ND		ND		ND	ND		ND	ND		ND		ND			ND	89.0			0.124±0.033

Notes: MESD: methanolic extract of Sterculia diversifolia; IC₅₀: half maximum inhibitory concentration; SEM: standard error of mean; DCM: dichloromethane; ND: Not given dose.

3.3.2. Lipoxygenase Inhibitory assay

The crude MESD (stem bark and leaves) and its fractions were screened for Lipoxygenase inhibitory activity. The maximum inhibitory activity of stem bark extract and fractions were observed for EtOAc (92.75%) with IC₅₀ value of (37.2 ± 0.30) µg/mL, followed by DCM (92.10%) with IC₅₀ value of (48.4 ± 0.17) µg/mL, crude MESD (88.50%) with IC₅₀ value of (54.6 ± 0.40) µg/mL, n-hexane (77.50%) with IC₅₀ value of (70.2 ± 0.33) µg/mL and n-butanol (61.75%) with IC₅₀ value of (98.30 ± 0.10) µg/mL respectively as shown in table 3. The maximum inhibitory activity of leaves extract and fractions were observed for DCM (94.88%) with IC₅₀ value of (40.08 ± 0.70) µg/mL, followed by EtOAc (94.55%) with IC₅₀ value of (42.80 ± 0.92) µg/mL, crude MESD (86.98%) with IC₅₀ value of (60.24 ± 0.32) µg/mL, n-hexane (71.44%) with IC₅₀ value of (80.84 ± 0.67) µg/mL respectively as shown in Table 3. The Lipoxygenase inhibitory activity of standard drugs (Baicalein) was 22.0 µM respectively.

3.3.3. β-glucuronidase inhibitory assay

The crude MESD (stem bark and leaves) and its fractions were screened for β-glucuronidase inhibitory activity. EtOAc fraction of stem bark showed maximum inhibitory activity (70.60%) with IC₅₀ value of (56.14 ± 0.26) µg/mL, while other fractions showed non-significant results. Similarly maximum inhibitory activity of leaves extract and fractions were observed for EtOAc (74.68%) with IC₅₀ value of (50.30 ± 0.64) µg/mL, while other fractions showed non-significant results as shown in Table 3. D-Saccharic acid 1, 4 lactone (standard drug) showed 89.4% inhibition with IC₅₀ values of (48.78 ± 2.16) µg/mL.

3.3.4. Urease inhibitory assay

Crude MESD and its fractions of both stem bark and leaves were screened for urease inhibitory activity. Among stem bark samples, EtOAc fraction showed significant inhbitory effect (83.67%) with IC₅₀ value of (116.4 ± 1.29) µg/mL against urease enzyme while mod-erate inhibitory activity was shown by the n-butanol fraction (55.33%) with IC₅₀ value of (458.24 ± 2.10) µg/mL respectively. Among leaves extract and fractions, significant inhibition was showed by ethyl acetate fraction (82.33%) with IC₅₀ value of (128.50 ± 1.08) µg/mL, while moderate inhibitory activity was shown by the n-butanol fraction (51.92%) with IC₅₀ value of (480.34 ± 1.56) µg/mL as shown in table 3. The percent inhibition for thiourea (standard drug) was 98.2 % with IC₅₀ values of (21.00 ± 0.01) µg/mL.

3.3.5. Xanthine oxidase inhibitory assay

Crude MESD and its various fractions of both stem bark and leaves were screened for xanthine oxidase inhibitory potential. Maximum inhibitory activity of stem bark extract and fractions were observed for DCM fraction (84.62%) with IC₅₀ value of (108.4 ± 0.88) µg/mL, followed by EtOAc fraction (76.34%) with IC₅₀ value of (194.28 ± 1.84) µg/mL respectively, while among leaves extract and fractions, significant inhibitory effect against urease was also shown by DCM fraction (89.20%) and EtOAc fraction (78.55%) with IC₅₀ value of (96.42 ± 1.80) and (190.32 ± 1.00) µg/mL. The percent inhibition for allopurinol (standard drug) was 98.6 % with IC₅₀ values of (2.00 ± 0.01) µg/mL.

3.3.6. Carbonic anhydrase inhibitory assay

The crude MESD and various fractions of stem bark and leaves were evaluated for carbonic anhydrase inhibitory assay (Table 3). Maximum inhibitory activity of stem bark extract and fractions were observed for EtOAc (78.28%) with IC₅₀ value of (182.28 ± 0.10) µg/mL, followed by crude MESD (60.60%) and DCM fraction (46.38%) with IC₅₀ value of (250.26 ± 0.40) and (495.82 ± 0.16) µg/mL respectively. Similarly maximum inhibitory activity of leaves extract and fractions were observed for EtOAc (83.48%) with IC₅₀ value of (154.26 ± 0.98) µg/mL, followed by crude MESD (68.24%), DCM fraction (59.32%) and n-butanol (49.04%) with IC₅₀ value of (206.02 ± 1.28), (406.70 ± 2.52) and (500.64 ± 0.90) µg/mL respectively.

4. DISCUSSION

Weed is one of the significant cause for huge economic losses all over the world by reducing the quality and quantity of agricultural crops. It is estimated that weeds causes a loss of about 12% costing to about US 33 billion dollars in United State, while in developing countries the situation is more worst.²⁵ There is a problem that resistance arises against these herbicides because of frequent use of these herbicides. The species have the affinity to convert into other species that have much similarity with beneficial plants. The haphazard use of herbicides also produce many environmental and health pollution problems, so there is an immense need of a herbicide showing significant pytotoxic activity with less toxicity to human health. It is evident from results that phytotoxic activity showed significant outcomes in dose dependant manner in both stem bark (ethyl acetate and n-butanol) and leaves (ethyl acetate, n-butanol and n-hexane) fractions. Literature showed that plants, their products and different isolated compounds possess phytotoxic potential, so they can be used as herbicide.²⁶ The phytotoxicity of the plant has proved that weeds could be controlled without any harmful effect on the crop growth as well as overall yield which results in significant increase in crops production.²⁷ On the basis of results it is therefore assumed that the phytotoxic agents of the stem bark and leaves could be a significant natural source for weeds control in a sustainable way for better production of crop.²⁸

Natural products are used as structural models for the novel molecules synthesis and exploited for their pharmacological properties. However, still there is not much in literature about studies on the natural products potential mutagenic and toxicological effects to understand the genotoxic effects of new phyto-therapeutic agents. It is crystal clear from the present study that DCM fraction showed significant genotoxic activity followed by ethyl acetate fraction. This activity is performed through comet assay. In recent times, comet assay grip special attention in order to identify substances with genotoxic effect. Comet assay is used for the primary DNA damage detection in individual cells. It is a very fast and sensitive tool for detection. Several classes of DNA injury could be detected with this method like single or double strand breaks, incomplete repair of a basic sites, alkali labile sites and cross links.²⁹ This activity showed a little increase in total damaged cells and scores in animal groups treated with the Sterculia diversifolia crude extracts and its fractions. The results are non-significant except DCM and ethyl acetate fractions at higher concentrations. It is known that many flavonoids and alkaloids have shown to be genotoxic in a variety of prokariotic and eukariotic cells and in vivo systems.^30,⇓-32 For genotoxicity, the mechanistic basis needs full elucidaion, although structure-activity relationship studies have identified as requisite flavonoids structural features.

Acetyl cholinesterease inhibitory activity showed significant results in both stem bark and leaves fractions. Acetylcholinesterase is an essential enzyme in the human body.³³ The biological role of AChE is the cessation of impulse transmissions in nervous system at cholinergic synapses via quick hydrolysis of neuro-transmitter acetylcholine.³⁴ Alzheimer's disease patient central cholinergic function is improved by AChE inhibitors application and as a result of this the deficiency in central nervous system functions is recompense. This is the only approved therapy.³⁵ Therefore, this is an important issue to search the natural products that having AChE inhibitory activity.

Lipoxygenase inhibition was shown by ethyl acetate, DCM, MESD and n-hexane fractions of stem bark and leaves. Lipoxygenase is a non-heme iron containing dioxygenase extensively dispersed in nature. Lipoxygenases enzymes converts linoleic, arachidonic and other polyunsaturated fatty acid into biologically active metabolites that are involved in immune and inflammatory responses. In human, lipoxygenase genes have been identified and due the diversity of lipoxygenase genes, the role of lipoxygenase is complex in the development and succession of cancer.³⁶

β-glucuronidase inhibitory activity showed highly significant results in ethyl acetate fraction of both stem bark and leaves. It is reported that β- glucuronidase activity increases in various diseases e.g. AIDS, arthritis, cancer and liver disorders.³⁷ Thus natural products which possess β-glucuronidase inhibition also possess cytotoxic effect. In living system glucuronidation leads to detoxification process.³⁸

Urease inhibitory activity showed highly significant results in ethyl acetate fraction of both stem bark and leaves. Urease catalyzes urea hydrolysis to ammonia and carbon dioxide. Ureas enzyme is pathogenic when upregulated, mediating ailments e.g. hepatic coma, peptic ulcer, pyelonephritis and urinary lithiasis. It also facilitates Proteus mirabilis, Helicobacter pylori and Yersinia enterocolitica infections. Urease increases the stomach pH which helps in Helicobacter pylori colony formation. This leads to the pathogenesis of peptic ulcer, gastritis and cancer.³⁹ Various extract of plants have shown urease inhibitory activity e.g. Aristolachia bracteata, Achillea millefolium, Ginkgo biloba, Taraxacum officinale, Diospyros lotus etc.^39,40

Xanthine oxidase inhibition was shown by DCM fraction of both stem bark and leaves. This enzyme plays an important role in humans in the metabolism of purine nucleotide which converts hypoxanthine into xanthine, which is further converted into uric acid. Uric acid excessive production leads to a pathological condition named gout, in which uric acid accumulation occurs in joints. This uric acid accumulation in joints causes inflammation and severe pain.⁴¹ The gout prevalence is higher in male (> 30 years of age) and female (> than 50 years of age). Allopurinol is used worldwide for this pathological condition.⁴² It is reported in the literature that plant extract exhibits its anti-gout activity through xanthine oxidase inhibition.

Carbonic anhydrase inhibitory activity showed highly significant results in ethyl acetate fraction of both stem bark and leaves. Carbonic anhydrase is involved in physiological process, related to transport and respiration of bicarbonate/CO₂ between metabolizing tissues and lungs.³⁹ It has been reported that this enzyme expression leads to colorectal cancer, glaucoma, leukemia, epilepsy and cystic fibrosis. Polyphenolic compounds isolated from plants have been reported as carbonic anhydrase inhibitors.⁴³ Both urease and carbonic anhydrase are key enzymes in the living organisms physiology. During homeostasis, urease elevate stomach pH, while carbonic anhydrase plays a vital role in acid-base balance, CO₂ and ion transport.

In the management of various pathological conditions, plant origin natural products are important. The enzymes inhibitory effect of these agents in human beings has got a special place in the field of drug discovery. Thus various pathological conditions could be treated by enzyme inhibitors.

In conclusion, this study emphasizes the important phytotoxic, genotoxic and enzyme inhibitory activity of Sterculia diversifolia stem bark and leaves. It is concluded that Sterculia diversifolia stem bark and leaves bears phytotoxic, genotoxic and enzyme inhibitory agents.