Evaluation of antihyperlipidemic and antitumor activities of isolated coumarins from Salvadora indica

Abstract

Context. Salvadora indica Wight (Salvadoraceae) contains a number of medically beneficial properties including abrasives, astringents and antiseptics. Traditionally, it was used by ancient Arabs to whiten and polish teeth.

Objective: This study explores the antihyperlipidemic and antitumor effects of an ethanol extract of S. indica and its isolated phytoconstituents in rodents.

Material and methods: Flash chromatography was used for the isolation of phytoconstituents from the stems of S. indica. An antihyperlipidemic study was carried out in Triton loaded rats. Animal groups were given intraperitoneal (i.p.) injection of Triton WR 1339 at dose of 400 mg/kg body weight (b.w.). Furthermore, antitumor activity was investigated in hybrid mice (of C57BL strain + Swiss albino strain). The animals were observed for tumor growth after injection of B16F10 melanoma cells into the dorsal skin of mice.

Results: The stems of S. indica yielded xanthotoxin and umbelliferone through chromatographic separation techniques. The structures of the compounds were elucidated by spectroscopic data interpretation and showed antihyperlipidemic activity. The study showed significant reduction in total cholesterol (TC) (p < 0.01), triglycerides (TGs) (p < 0.001), low-density lipoproteins (p < 0.01) level whereas increased in high-density lipoprotein (p < 0.01) at a significant level, after the treatment. Pretreatment with the extract and phytoconstituents also showed delayed tumor growth by increasing the volume doubling time (VDT) (p < 0.01), growth delay (GD) (p < 0.01) and mean survival time (p < 0.001).

Discussion and conclusion: Acute treatment caused a stimulatory effect on high density lipoprotein level and inhibition in TC and TG elevation induced by Triton. Tumor regression studies showed a regression response for tumor growth in vivo of murine mouse melanoma as demonstrated by increasing the VDT and GD.

• Total cholesterol
• umbelliferone
• xanthotoxin

Introduction

Salvadora indica Wight, Salvadoraceae, (syn. Salvadora persica), is a small tree or shrub with a crooked trunk. It is reported to exhibit antiulcer (Monforte et al., 2001), anticonvulsant (Monforte et al., 2002), analgesic (Mansour et al., 1996), antibacterial (Al-Ali & Al-Lafi, 2003; Sofrata et al., 2008), antiinflammatory (Al-Hindwai et al., 1989) and antioxidant activities (Dhankhar et al., 2012). It contains a number of medically beneficial properties including abrasives, astringents and antiseptics. Its main constituents are trimethylamine, an alkaloid which may effectively be salvadorine, chlorides, sulfur, terpenes, vitamin C, glycosides, large amounts of fluoride and silica, small amounts of tannins, saponins and flavonoids (Asolkar et al., 1992; Bungorn et al., 2001; Chatterjee, 1990). Small sticks have been used as toothpicks (Ezoddini-Ardakani, 2010).

The leaves, stem bark and root of S. indica have a long history of medicinal uses. The fruit is sweet, aphrodisiac, stomachic, improves appetite and useful in biliousness. The leaves are bitter, corrective, deobstruent, astringent to the bowels, tonic to the liver, diuretic, analgesic, anthelmintic and useful in nose troubles, piles, scabies, leucoderma, lessen inflammation and strengthen the teeth.

Hyperlipidemia is a general term; it could be either high cholesterol in the blood (hypercholesterolemia), high triglycerides (TGs) in the blood (hypertriglyceridemia) or be both. High plasma lipid levels, mainly total cholesterol (TC), TGs and low density lipoproteins (LDL) along with decrease in high density lipoprotein (HDL) are known to cause hyperlipidemia, which is core in initiation and progression of arteriosclerosis impasse. Therefore, prime consideration in therapy for hyperlipidemia and arteriosclerosis is to enervate the elevated plasma level of TC, TG and LDL along with increase in HDL lipid levels.

The production of new cells is so regulated that the numbers of any particular type of cell remain constant. Under abnormal circumstances, these cells give rise to clones of cells that can expand to a considerable size, producing a tumor or neoplasm (Barker et al., 1980; Thomlinson & Gray, 1955).

This article also deals with the isolation and characterization of coumarin derivatives. Coumarin is a fragrant organic chemical compound in the benzopyrone chemical class. Dietary exposure to benzopyrones is quite significant, as these compounds are found in vegetables, fruit, seeds, nuts, coffee, tea and wine (Bruneton, 1999).

Coumarins are important compounds that contribute to the adaptation of plants to biotic or abiotic stresses. Coumarin and its derivatives are considered as phenylpropanoids (Kulkarni et al., 2006). Coumarins are reported to exhibit anticonvulsant (Luszczki et al., 2010), antihyperglycemic (Ramesh & Pugalendi, 2006), antimyotoxic (Toyama et al., 2011), antiinflammatory and antiviral activities (Selim & Ouf, 2012).

This study was designed to investigate and to explore the potential of S. indica in the treatment of hyperlipidemia and tumors.

Material and methods

Plant material

The stems of S. indica were collected in the month of July 2010 from Bhopal (Madhya Pradesh), India, and were identified and authenticated by Dr. Zia ul Hassan, Assistant professor, Department of Botany, Saifia College of Science & Education, Bhopal. A voucher specimen no. 175/Bot/Safia/2010 was deposited in the herbarium of the Botany Department.

Extraction and fractionation

Dried drug (800 g) was taken and coarsely powdered and then exhaustively extracted with 90% ethanol in a Soxhlet apparatus (VNS Institute of Pharmacy, Bhopal, MP, India). The ethanol extract so obtained was freed of solvent under vacuum to yield 74 g (9.25% yield) of dark brown mass. The solvent-free ethanol extract was further dissolved and extracted with chloroform. The chloroform layer was separated and allowed to evaporate. Ethanol extract and chloroform fraction were thus obtained.

The chloroform fraction 48.6 g (7.08% yield) was subjected to further study involving isolation of active components. Qualitative chemical tests were performed to assess the presence of various phytoconstituents. The tests revealed the presence of sterols, tannins, coumarins, alkaloids and glycosides in the ethanol extract of S. indica.

Instrumentation used

Flash chromatography was done using a Buchi controller C-610 apparatus (NIPER, Mohali, India). The ultraviolet (UV) spectra were recorded on a spectrometer by Shimadzu UV 1700 model in MeOH/EtOH (VNS Institute of Pharmacy, Bhopal, MP, India). The infrared (IR) spectra were measured on a Jasco FT/IR-5300 spectrophotometer (RGTU, Bhopal, MP, India). The ¹H spectra and ¹³C spectra were recorded on a V 300 and BKS nuclear magnetic resonance (NMR) spectrometer (NIPER) using dimethyl sulfoxide (DMSO) and CDCl₃ as solvents. Mass spectra were recorded on a Scan AP spectrometer/JEOL JMS AX-500 spectrometer (RGTU, Bhopal, MP, India).

Chromatographic isolation of compounds

Further elution of the column was done using flash chromatography. Elution of the column with chloroform (fractions 60–85) yielded crystalline powder.

The powder was further adsorbed on silica gel (60–120 mesh). It was dried, packed and chromatographed over a silica gel column packed in petroleum ether. The column was eluted with petroleum ether, chloroform and methanol successively in order of increasing polarity to isolate the active constituents.

Elution of the column with chloroform (fraction 11–16) yielded white needle-shaped crystals recrystallized from ethanol. The yield was found to be 533.10 mg (0.176%). Thin layer chromatography (TLC) of the powdered sample was carried out using petroleum ether:chloroform:ethanol:acetic acid (5:1:0.5:0.5). This solvent system gave the best resolution.

Further elution of the column with chloroform:methanol (120:80) (fraction 29–40) yielded creamish yellow amorphous powder recrystallized from ethanol. The powder was further adsorbed on silica gel (60–120 mesh). It was dried, packed and chromatographed over a silica gel column packed in petroleum ether. The column was eluted with petroleum ether, chloroform and methanol successively in order to increase polarity to isolate the active constituents.

Elution of the column with chloroform:methanol (60:40) (fraction 13–20) yielded creamish white crystals recrystallized from ethanol. The yield was found to be 459.74 mg (0.153%). TLC of the powdered sample was carried out using various solvent systems. The appropriate one was found to be chloroform:n-butanol:ethanol:acetic acid (2:4:1:1). This solvent system gave the best resolution.

Characterization of compounds

Compound I has Retention factor (Rf): 0.53; melting point (m.p.): 146–148 °C; λmax in EtOH: 212, 226, 254 and 312 nm; IR bands (KBr): 2854, 1733, 1590, 1143 and 1326 cm⁻¹; ¹H-NMR (CDCl₃): δ 4.28 (3H, s, CH₃O), 6.29 (1H, d, H-6, J = 9.8 Hz), 7.27 (1H, d, H-4), 7.13 (1H, d, H-3), 7.60 (1H, d, H-2, J = 2.4 Hz) and 8.17 (1H, s, H-5); ¹³C-NMR (CDCl₃): δ 161.26 (C-2), 93.82 (C-3), 144.79 (C-4), 112.63 (C-5), 106.37 (C-6), 149.56 (C-7), 139.29 (C-9), 158.37 (C-9a), 152.69 (C-8a), 105.05 (C-3a), 77.02 (C-4a) and 60.08 (9-OCH₃); and MS m/z: 216 [M–H]⁻ (C₁₂H₈O₄), 201, 176, 148, 91 and 67.

Compound II has R_f: 0.47; m.p: 227–231 °C; λmax in EtOH: 324 nm; IR bands (KBr): 3644, 2854, 1814, 1670, 1442, 1053, 931 and 840 cm⁻¹; ¹H-NMR (DMSO): δ 10.58 (1H, s, OH-7), 7.95 (1H, d, H-4, J = 9.3 Hz), 7.55 (1H, d, H-5, J = 8.7 Hz), 6.23 (1H, d, H-3, J = 10.32 Hz), 6.74 (1H, d, H-8), 6.83 (1H, dd, H-6, J = 2.7 Hz); ¹³C-NMR (DMSO): δ 160.52 (C-2), 155.56 (C-6), 144.55 (C-4), 129.74 (C-8), 113.17 (C-3), 111.46 (C-5), 111.33(C-7), 102.23 (C-4a) and 161.35 (C-8a); MS m/z: 162 [M--H]^– (C₉H₆O₃), 77, 89, 105, 117 and 133.

Screening for hypolipidemic activity

Screening for hypolipidemic activity was carried out in male albino rats weighing 100–120 g. Triton WR 1339 (Tyloxapol) was purchased from Sigma Chemicals Co., St. Louis, MO.

Preparation of test material

Ethanol extract, xanthotoxin and umbelliferone were suspended in distilled water plus polyoxyethylene sorbiton monooleate (Tween 80). Triton was dissolved in normal saline to give a 7% solution.

Compound I. Xanthotoxin.

Compound II. Umbelliferone.

Animal model

The albino rats were selected and housed in polypropylene cages maintained under controlled conditions. The animals were fed standard food and acidified water ad libitum. Male albino rats of 6–8 weeks old and weighing 100–120 g were taken for the experiments. The usage of animals was approved by the ethical committee of the Research Centre having following committee for the purpose of control and supervision on experiments on animals (CPCSEA) Reg. No. 778/03/c/CPCSEA.

Measurement of biochemical parameters

Albino rats were divided into six groups of six rats each. Group I served as vehicle control. Group II was kept as hyperlipidemic group and animals were administered with Triton only. Group III was kept as standard. Groups IV, V and VI were treated with ethanol extract, xanthotoxin and umbelliferone, respectively. Group II–VI were given i.p. injection of Triton WR 1339 at dose of 400 mg/kg b.w. After 24 h of Triton administration, animals in Groups III, IV, V and VI were treated with standard (β-sitosterol), ethanol extract, xanthotoxin and umbelliferone, respectively, at the oral dose of 200 mg/kg. The treatment was continued for 5 days with a view to see the effect on lipid profile.

The blood samples were withdrawn from the eye vein, 1 h after the last dose of drug administration, and transferred directly into centrifuge tubes and allowed to clot at room temperature for 20–25 min and centrifuged for 15–20 min at 2000 rpm. The supernatant clear serum thus obtained was transferred carefully with the help of a micropipette into small test tubes for estimation. The serum concentration of TC, HDL and TG were measured by standard procedures using an auto-analyzer (Friedman & Bayer, 1957).

Histopathological study

Histopathological study of rat liver of all the six groups was also performed. Tissue sections of 3 μm thickness were stained with Ehrlich’s hematoxylin and eosin and examined by light microscopy.

Screening for antitumor activity

Animal model

The hybrid mice (of C57BL strain + Swiss albino strain) were selected from a breed colony maintained in the animal house of the Research Department of Jawaharlal Nehru Cancer Hospital, Bhopal, MP, India. The mice were housed in polypropylene cages maintained under controlled conditions. The animals were fed standard feed (formula obtained from the Cancer Research Institute, Mumbai, India) and water ad libitum. Mice of 6–8 weeks old and weighing 25–30 g were selected from the above colony for the experiments.

Tumor model: B16F10 melanoma

B16F10 melanoma originally obtained from the National Cell Centre, Pune, India, was maintained by serial transplantation in C57BL mouse.

Tumor propagation

Tumor-bearing mouse was sacrificed by cervical dislocation, and the whole animal was dipped in 70% alcohol, and the tumor was excised to single cell suspension by mechanical dispersion. The cell suspension was filtered through a 45 µ nylon mesh. The single cell suspension was then passed through different gauge size needles. The cell suspension was again passed through nylon mesh so as to remove the clumps of cells.

Methodology

The mice were divided into four groups. Group I served as control. Groups II, III and IV were injected daily with the ethanol extract, xanthotoxin and umbelliferone 50 mg/kg b.w. i.p. for 10 consecutive days. Three weeks after the last injection of the test compounds, the animals were injected subcutaneously with 5 × 10⁵ viable B16F10 melanoma cells into the dorsal skin. The animals were observed for the growth of tumor. Volume doubling time (VDT) and growth delay (GD) were calculated (Uma Devi & Guruprasad, 2001; Uma Devi et al., 2001).

Tumor growth kinetics

The tumor size was measured every alternate day, and the tumor volume was calculated. Tumor diameters are measured with digital callipers, and the tumor volume in mm³ is calculated by the formula: volume = (width)² × length/2.

Tumor growth response was assessed from the following parameters:

• VDT – The time, in days, for the tumor’s size to reach double the treatment volume.

• GD – Difference in the time, in days, needed for the treated and untreated tumor to reach five times the treatment volume.

Statistical analysis

Statistical evaluation of the data was done by Student’s t-test (Graph PAD Instat software, Kyplot (VNS Institute of Pharmacy, Bhopal, MP, India)). A value of p < 0.05 was considered to be significant.

Results

Preliminary phytochemical screening revealed the presence of sterols, tannins, coumarins, alkaloids and glycosides in the ethanol extract of S. indica.

The structure of the compound obtained from chloroform fraction of S. indica was elucidated as xanthotoxin and umbelliferone on the basis of spectral data analysis. The purity of xanthotoxin and umbelliferone was also confirmed by spectroscopic studies.

Compound I (xanthotoxin) was obtained as white needle-shaped crystals from chloroform eluent. Its IR spectrum displayed characteristic absorption bands at 2854 cm⁻¹ (C--H), 1733 cm⁻¹ (C=O), 1143 (C--O stretching) and 1326 cm⁻¹ (α, β-unsaturated lactone). The fast atom bombardment (FAB) mass spectrum of the compound exhibited a molecular ion peak at m/z 216 (also base peak) consistent with the molecular formula (C₁₂H₈O₄). The important peaks appearing are 201, 176, 148, 91 and 67. Moreover, the peak obtained at m/z 201 is due to the cleavage of a methoxy moiety in the molecule. The ¹H NMR spectrum of the compound suggested a characteristic signal for a methoxy group at δ 4.28 as singlet. The spectrum showed two proton doublets at δ 7.13 for H-3 and δ 7.27 for H-4. Moreover, two proton doublets were appeared at δ 6.29 (1H, d, H-6, J = 9.8 Hz), 7.60 (1H, d, H-2, J = 2.4 Hz). The ¹³C-NMR spectrum of the compound suggested positions of carbons at δ 161.26 (C-2), 93.82 (C-3), 144.79 (C-4), 112.63 (C-5), 106.37(C-6), 149.56 (C-7), 139.29 (C-9), 158.37 (C-9a), 152.69 (C-1a), 105.05 (C-4a), 77.02 (C-5a) and 60.08 (9-OCH₃). After comparing the data with spectral information from the literature, the first component was confirmed as xanthotoxin.

Compound II (umbelliferone) was obtained as creamish white crystals from the chloroform:methanol (60:40) eluent. Its IR spectrum displayed characteristic absorption bands for 3644 cm^–1 (O--H), 2854 cm⁻¹ (C--H), 1814 cm⁻¹ (C=O), 1670 (α, β-unsaturation), 1053 cm⁻¹(C--O) and 840 cm⁻¹ (C--H bending). The FAB mass spectrum of the compound exhibited a molecular ion peak at m/z 162 consistent with the molecular formula (C₉H₆O₃). The important peaks appearing are 77, 89, 105, 117 and 133. It showed a strong molecular ion peak at m/z 162 (80%) and base peak at m/z 133 due to loss of carbon monoxide. The ¹H NMR spectrum of the compound suggested a characteristic signal for a hydroxyl group at δ 10.58 as singlet. The spectrum showed one proton doublet at δ 6.74 for H-8 and a double doublet at δ 6.83 for H-6 (J = 2.7 Hz). Moreover, further three proton doublets appeared at 7.95 (1H, d, H-4, J = 9.3 Hz), 7.55 (1H, d, H-5, J = 8.7 Hz) and 6.23 (1H, d, H-3, J = 10.32 Hz). The ¹³C-NMR spectrum of the compound suggested positions of carbons at δ 160.52 (C-2), 155.56 (C-6), 144.55 (C-4), 129.74 (C-8), 113.17 (C-3), 111.46 (C-5), 111.33(C-7), 102.23 (C-4a) and 161.35 (C-8a). After comparing the data with spectral information from the literature, the second component was confirmed as umbelliferone.

Triton WR-1339 acts as a surfactant and suppresses the action of lipases to block the uptake of lipoproteins from circulation by extrahepatic tissues, resulting in increased blood lipid concentration (Friedman & Bayer, 1957). The possible mechanism of activity may be due to enhancement of the activity of lecithin acyl transferase (LCAT) and inhibition of the action of hepatic TG-lipase on HDL (Patil & Dixit, 2004).

The rats treated with Triton showed an increase in serum cholesterol level as compared to initial level. In the S. indica ethanol extract-treated group, the initial level of TC was found to be 56.51 ± 0.95 mg/dl, which was increased up to 75.02 ± 1.03 mg/dl by Triton. After the treatment with drug extract, the TC level was reduced to 66.8 ± 0.29 mg/dl. In the xanthotoxin-treated group, the initial level of TC was 59.29 ± 0.61 mg/dl, which was increased up to 72.55 ±1.15 mg/dl; and after the treatment with test drug, the TC level was reduced to 64.94 ± 0.92 mg/dl. In the umbelliferone-treated group, the initial level of TC was found to be 60.44 ± 0.88 mg/dl, which was increased up to 73.42 ±0.96 mg/dl by Triton. After the treatment with test drug, the TC level was reduced to 65.30 ± 0.62 mg/dl (Table 1).

Table 1. Effect of ethanol extract, xanthotoxin and umbelliferone from Salvadora indica on total cholesterol level (mg/dl) in the Triton-induced hyperlipidemic model.

Group	Initial	Total cholesterol level after the administration of Triton	Total cholesterol level the after 1 h of the last dose of vehicle/ drug treatment
Control	59.57 ± 0.58	71.52 ± 0.93	73.60 ± 0.54
Hyperlipidemic	59.74 ± 0.76	74.11 ± 0.54	85.97 ± 0.77
Standard (β-sitosterol)	57.60 ± 0.63	71.75 ± 0.33	58.80 ± 1.13
S. indica ethanol extract	56.51 ± 0.95	75.02 ± 1.03	66.80 ± 0.29^a
Xanthotoxin	59.29 ± 0.61	72.55 ± 1.15	64.94 ± 0.92^a
Umbelliferone	60.44 ± 0.88	73.42 ± 0.96	65.30 ± 0.62^a

Total cholesterol concentrations are estimated by standard method. Values are expressed as mean ± SEM for six animals in each group.

^ap < 0.01 compared with hyperlipidemic group.

In the S. indica ethanol extract-treated group, the initial level of TG was 54.79 ± 0.79 mg/dl, which was increased up to 89.46 ± 1.59 mg/dl; and after treatment with the test drug, it was decreased to 80.27 ± 0.27 mg/dl. In the xanthotoxin-treated group, the initial level of TG was found to be 58.94 ±0.23 mg/dl, which was increased up to 90.63 ± 0.90 mg/dl by Triton. After the treatment with sample, the TG level was reduced to 77.81 ± 0.42 mg/dl. In the umbelliferone-treated group, the initial levels of TG was 55.15 ± 1.15 mg/dl, which was increased up to 82.47 ± 1.07 mg/dl; and after treatment with the test drug, the TG was decreased to 78.26 ± 0.54 mg/dl (Table 2).

Table 2. Effect of ethanol extract, xanthotoxin and umbelliferone from Salvadora indica on triglyceride level (mg/dl) in Triton-induced hyperlipidemic model.

Group	Initial	Triglyceride level after the administration of Triton	Triglyceride level after 1 h of the last dose of vehicle/ drug treatment
Control	58.85 ± 0.53	92.67 ± 0.35	101.30 ± 1.61
Hyperlipidemic	57.60 ± 0.31	84.10 ± 0.88	135.80 ± 1.95
Standard (β-sitosterol)	55.34 ± 0.78	83.58 ± 0.53	72.82 ± 0.82^a
S. indica ethanol extract	54.79 ± 0.79	89.46 ± 1.59	80.27 ± 0.27^a
Xanthotoxin	58.94 ± 0.23	90.63 ± 0.90	77.81 ± 0.42^a
Umbelliferone	55.15 ± 1.15	82.47 ± 1.07	78.26 ± 0.54^a

Triglyceride concentrations are estimated by standard method. Values are expressed as mean ± SEM for six animals in each group.

^ap < 0.001 compared with hyperlipidemic group.

In the S. indica ethanol extract-treated group, the initial level of HDL was 51.79 ± 0.58 mg/dl, which was increased up to 52.03 ± 0.64 mg/dl; and after the treatment with the test drug, it was increased up to 54.46 ± 0.51 mg/dl. In the xanthotoxin-treated group, the initial level of HDL was found to be 51.74 ± 0.23 mg/dl, which was increased up to 53.88 ±1.05 mg/dl by Triton. After the treatment with sample, the HDL level was raised up to 56.83 ± 0.26 mg/dl. In the umbelliferone-treated group, the initial levels of HDL was 52.33 ±1.28 mg/dl, which was increased up to 54.32 ± 0.92 mg/dl; and after the treatment with the test drug, the HDL was increased up to 56.49 ± 1.11 mg/dl (Table 3).

Table 3. Effect of ethanol extract, xanthotoxin and umbelliferone from Salvadora indica on HDL level (mg/dl) in Triton-induced hyperlipidemic model.

Group	Initial	HDL level after the administration of Triton	HDL level after 1 h of the last dose of vehicle/ drug treatment
Control	53.12 ± 0.47	53.87 ± 0.64	53.92 ± 0.56
Hyperlipidemic	52.24 ± 0.31	52.60 ± 0.38	53.63 ± 0.93
Standard (β-sitosterol)	55.34 ± 0.78	56.22 ± 0.46	58.83 ± 0.38^a
S. indica ethanol extract	51.79 ± 0.58	52.03 ± 0.64	54.46 ± 0.51
Xanthotoxin	51.74 ± 0.23	53.88 ± 1.05	56.83 ± 0.26^a
Umbelliferone	52.33 ± 1.28	54.32 ± 0.92	56.49 ± 1.11^a

HDL concentrations are estimated by standard method. Values are expressed as mean ± SEM for six animals in each group.

^ap < 0.01 compared with hyperlipidemic group.

In the S. indica ethanol extract-treated group, the initial level of LDL was found to be 15.97 ± 0.21 mg/dl, which was increased up to 42.79 ± 0.69 mg/dl by Triton. After the treatment with sample, the LDL level was reduced to 40.53 ± 0.81 mg/dl. In the xanthotoxin-treated group, the initial level of LDL was 13.11 ± 0.36 mg/dl, which was increased up to 39.86 ± 0.59 mg/dl; and after the treatment with test drug, the LDL level was reduced to 34.20 ± 0.80 mg/dl. In the umbelliferone-treated group, the initial level of LDL was found to be 14.09 ± 0.73 mg/dl, which was increased up to 38.44 ± 1.02 mg/dl by Triton. After the treatment with the test drug, the LDL was reduced to 36.76 ± 1.39 mg/dl (Table 4).

Table 4. Effect of ethanol extract, xanthotoxin and umbelliferone from Salvadora indica on LDL level (mg/dl) in Triton-induced hyperlipidemic model.

Group	Initial	LDL level after the administration of Triton	LDL level after 1 h of the last dose of vehicle/ drug treatment
Control	18.22 ± 0.29	36.18 ± 0.53	39.95 ± 0.85
Hyperlipidemic	13.66 ± 0.25	33.69 ± 0.50	58.50 ± 0.47
Standard (β-sitosterol)	13.32 ± 0.16	30.24 ± 0.59	27.56 ± 0.41^a
S. indica ethanol extract	15.97 ± 0.21	42.79 ± 0.69	40.53 ± 0.81^a
Xanthotoxin	13.11 ± 0.36	39.86 ± 0.59	34.20 ± 0.80^a
Umbelliferone	14.09 ± 0.73	38.44 ± 1.02	36.76 ± 1.39^a

LDL concentrations are estimated by standard method. Values are expressed as mean ± SEM for six animals in each group.

^ap < 0.01 compared with hyperlipidemic group.

As is evident from Figure 1(A), the histopathological alterations viz steatosis, sinusoidal dilatation and congestion, Kupfer cell hyperplasia or portal triaditis were not present in normal rat liver. Histopathology of liver in Triton-induced hyperlipidemic rats (Figure 1B) showed microvesicular steatosis along with sinusoidal dilatation, congestion and Kupfer cell hyperplasia. Liver histopathology (Figure 1C) of rats treated with Triton and standard showed only mild sinusoidal congestion. Portal tracts and overall hepatic architecture were within normal limits. Histopathology of liver in ethanol extract-treated rats (Figure 1D) showed mild vascular congestion and steatosis. Rats treated with xanthotoxin and umbelliferone (Figure 2A and B) showed mild feathery degenerative changes and mild vascular congestion but no steatosis. The overall hepatic architecture was maintained.

Figure 1. (A) Liver section of control rat. (B) Liver section of rat treated with Triton. (C) Liver section of rat treated with Standard drug. (D) Liver section of rat treated with S. indica ethanolic extract.

Figure 2. (A) Liver section of rat treated with xanthotoxin. (B) Liver section of rat treated with umbelliferone.

From the growth curves of tumor (Figure 3), it is clear that control tumors showed an exponential growth. The xanthotoxin and umbelliferone-treated groups produced slow growth response when compared to control. The VDT and GD were calculated from the growth curves of individual tumor-bearing mice.

Figure 3. Growth curve: effect of treatment on tumor growth in mice.

The silent period (i.e., time taken for palpable growth) for the control group was found to be one day, while in case of xanthotoxin- and umbelliferone-treated groups, it was found to be nine and five days, respectively, which was very significant (p < 0.0001) (Table 5).

Table 5. Response of B16F10 mouse melanoma to the treatment with ethanol extract of S. indica, xanthotoxin and umbelliferone at the dose of 50 mg/kg.

Parameter	Control	Ethanol extract	Xanthotoxin	Umbelliferone
Silent period	01 ± 0.34	03 ± 0.44	09 ± 0.63	05 ± 0.40^a
Volume doubling time (VDT), days	02 ± 0.28	03 ± 0.52	04 ± 0.17	03 ± 0.28^b
Growth delay (GD), days	–	01 ± 0.20	03 ± 0.51	02 ± 0.37^c
Mean survival time (MST)	24 ± 0.98	26 ± 0.67	32 ± 0.43	29 ± 0.22^c

Silent period, VDT, GD and MST.

Values are expressed as mean ± S.E.M. for six animals in each group.

^ap < 0.0001; ^bp < 0.01 compared to control; ^cp < 0.001.

The VDT observed for xanthotoxin- and umbelliferone-treated groups was found to be four and three days that was significant (p < 0.01) compared to control (Table 5).

The maximum survival time (MST) was observed to be 26 d for the control group. The MST observed for xanthotoxin- and umbelliferone-treated groups was 32 and 29 days, respectively, which was 8 and 5 days more than the control group (Table 5). Comparison in between MST of control group with xanthotoxin- and umbelliferone-treated groups was found to be significant (p < 0.001) (Table 5).

The GD was three days in xanthotoxin and two days in umbelliferone-treated groups, which was significant (p < 0.01) compared to control (Table 5).

Discussion

Research on herbal medicines is gaining ground, and the demand to use natural products in the treatment of various disorders is increasing worldwide. Investigations on herbal products might lead to the development of alternative drugs and strategies. Such alternative strategies are required for the effective management of hyperlipidemic disorders. LCAT plays a key role in the incorporation of free cholesterol into HDL and transferring it back to very LDL and LDL, which are taken back later in liver cells (Ghule et al., 2006). Moreover, alteration in cholesterol metabolism has been associated with the etiology of most human diseases. It is widely reported that hypercholesterolemia occasioned by a defect in cholesterol transportation, biosynthesis or catabolism is a risk factor in coronary heart disease (CHD) and atherosclerosis (Mohammed et al., 2011; Singh et al., 2010).

Recently, a number of clinical studies suggest that the increased risk of CHD is associated with a high serum concentration of TC, LDL-cholesterol (LDL-C) and TG. The abnormally high concentration of serum lipids is mainly due to the increase in the mobilization of free fatty acids from the peripheral depots (Ahmed et al., 2001). On the other hand, low serum concentration of HDL-C is also responsible for CHD (Parab & Mengi, 2002). Preclinical observations demonstrate that hyperlipidemia promotes accumulation of oxidatively modified low density lipoproteins in the arterial wall, promoting endothelial dysfunction and development of atherosclerosis and congestive heart diseases (Aikawa & Libby, 2004).

It is well known that LDL plays an important role in arteriosclerosis and that hypercholesterolemia is associated with a defect relating to the lack of LDL receptors. The decrease of cholesterol and LDL levels, achieved by administration of test samples, demonstrates a possible protection against hypercholesterolemia and the harm this condition brings about (Takahashi, et al., 2005).

It is also known that HDL-cholesterol levels have a protective role in coronary artery disease. Similarly, increased level of serum LDL-C results in increased risk for the development of atherosclerosis. The increased level of HDL cholesterol and decreased cholesterol level along with its LDL fraction, which is evident from the results, could be due to an increased cholesterol excretion and decreased cholesterol absorption through gastro-intestinal tract (Patil & Dixit, 2004).

The extract, fraction and the active phytoconstituent, inhibited the TC, TGs, LDL level and significantly increased HDL level. The active ingredients present in S. indica may recover the disorders in lipid metabolism noted in hyperlipidemic state.

Tumor regression studies showed a regression response for tumor growth in vivo of a murine mouse melanoma. The treatment produced a delay in tumor growth, as demonstrated by increasing the VDT and GD. Tumor regression was related to the immune-stimulatory properties of the antibody (Hardy et al., 1997). Indications are available that this plant has antioxidant properties. Oxidative stress has been implicated in numerous pathophysiological conditions including cancer. Prevention of oxidative damage can be employed as one of the ways in tumor regression.

Conclusion

This work characterized active phytoconstituents exhibiting antihyperlipidemic and antitumor potential from the stems of S. indica. CHDs are the clinical manifestation of atherosclerosis. Development of hyperlipidemia disease is a complicated process involving accumulation of lipid containing particles in the walls of coronary arteries. Treatment with ethanol extract of S. indica showed significant decreases in TG. HDL is considered to be a beneficial lipoprotein as it has an inhibitory effect in the pathogenesis of atherosclerosis. Low level of HDL is associated with high risk of coronary artery disease. In this investigation, treatment with ethanol extract and isolated phytoconstituent significantly decreased TC and TG level in serum, while HDL level in serum was significantly increased. Moreover, treatment with S. indica extract and phytoconstituents also showed a tumor regression response. These investigations may be quite useful as this drug is highly valued in the traditional system of medicine.

Declaration of interest

The authors report no conflicts of interest. The authors alone are responsible for the content and writing of this article.