CHEMICAL AND BIOLOGICAL STUDIES OF RUSSELIA EQUISETIFORMIS (SCH.&CHAM.) AERIAL PARTS
Eman M. Ahmed, Samer Y. Desoukey, Mostafa A. Fouad*, Mohamed S. Kamel
Department of Pharmacognosy, Faculty of Pharmacy, Minia University, Minia, 61519 Egypt
Keywords: Russelia equisetiformis, Chemical features, Bio-activity, Aerial parts
Abstract

From the aerial parts of Russelia equisetiformis, eight compounds were isolated (1–8) and identified by different spectral techniques as well as comparison with reported data or authentic samples. The isolated compounds included an alkane (n-pentatriacontane) (1), a mixture of ß-sitosterol and stigmasterol (2), a triterpene (lupeol) (3), a mixture of ß-sitosterol- and stigmasterol-1-O-ß-D-glucopyranosides (4), a phenylpropanoidglucoside (4-Allyl-2,6-dimethoxyphenol-1-O-ß-D-glucopyranoside(5), a flavonoid (vitexin) (6) and the two closely related phenylethanoids (acteoside) (7) and isoacteoside (8). The anti-inflammatory, antipyretic and antihyperglycemic activities were carried out on different plant fractions.

Article Information

Identifiers and Pagination:
Year:2016
Volume:8
First Page:23
Last Page:33
Publisher Id:JAppPharm (2016 ). 8. 23-33
Article History:
Received:November 27, 2015
Accepted:January 12, 2016
Collection year:2016
First Published:April 1, 2016

1.Introduction

Russelia equisetiformis Schlechtendalet Chamisso. (Russelia juncea Zucc.) belonging to family scrophulariaceae, recently introduced into the new family Plantaginaceae is native to Tropical South America especially in Mexico1-3. It is an evergreen, perennial, weeping shrub with attractive looking, green arching stems and tubular red blossoms, commonly named as fire cracker plant, coral plant, coral blow and fountain plant4. R. equisetiformis is traditionally used in Nigeria to cure malaria, cancer, inflammatory disorders, diabetes, leukemia, and in hair preparations to promote hair growth5-6. In Colombia, the fresh entire plant decoction is taken orally to cure kidney stones7 , while the whole plant and its aerial infusion is utilized as a complementary therapy for DM2 patients in Mexico8-9. An anti-oxidant, anti-inflammatory6, antinociceptive and analgesic properties10-11 were observed for different extracts of R. equisetiformis, together with antibacterial12, antimicrobial, cytotoxic13, CNS depressant14, hepatic functions activity5, hair growth promoter15 and membrane stabilizing activities16. The present study reported the separation and structure elucidation of eight compounds (Fig 1) from different fractions of the aerial parts of R. equisetiformis, in addition to evaluate the LD50, anti-inflammatory, antipyretic and antihyperglycemic activities of it. 

2. Materials and Methods

2.1 General

The EI-MS was measured on JEOL, JMS 600 Hz (Japan). 1D NMR was measured on both (JEOL JNM-LA 400 MHz and BRÜKER 600 MHz NMR spectrometers, Japan and Germany, respectively). Column chromatography (CC) was performed using silica gel (60-120 µm mesh size, Nile chemicals, Egypt) and sephadex LH-20 (25-100 mm mesh size, GE Health care, Sweden). TLC was carried on pre-coated silica gel plates (G60F254, E-Merck and FLUKA, Germany). The plates were examined under UV light via a portable UV lamp at ?max 365 and 254 nm (UVP, USA ) and visualized by spraying with10% v/v H2SO4 in ethyl acetate, then allowed to dry at room temperature followed by heating at 110–140°C  using a circulating hot-air oven (CARBOLITE, Germany) for 1–2 min. The following solvent systems were used for TLC: (I): n-Hexane-EtOAc (8.5:1.5 v/v), (II): n-Hexane-EtOAc (9:1 v/v), (III): CHCl3-MeOH (9.2:0.8), (IV):CHCl3-MeOH (9:1), (V):CHCl3-MeOH-H2O (8.5:1.5:0.1), (VI):EtOAc-MeOH (7:3). Authentic reference materials (ß-Sitosterol and stigmasterol) were obtained from Pharmacognosy department, Faculty of Pharmacy, Minia University, Minia, Egypt. For biological studies, a vernier caliber, digital thermometer and a glucometer (One Touch Horizon, LIFESCAN, Johnson and Johnson, Ltd) were used.

2.2 Plant Material

Aerial parts of R. equisetiformis were collected in February 2008 from El-Zohria garden, Cairo, Egypt. The plant was identified by Dr. Mamdoh Shokry, director of El-Zohria garden. A voucher specimen was deposited in the herbarium of the Pharmacognosy department, Faculty of Pharmacy, Minia University under the number (REMIN08).

2.3 Extraction and Isolation

Dried powdered aerial parts of R. equisetiformis (1.5 Kg) were extracted with 70% aqueous MeOH and concentrated to dryness in a rotary evaporator. The dried methanolic extract (A) was suspended in water and successively partitioned with three organic solvents to obtain n-hexane (11 gm) (B), chloroform (9 g) (C) and ethyl acetate (22.9 g) (D) fractions, together with the remaining concentrated aqueous fraction (E). A part of the n-hexane fraction (9 g) was subjected to silica gel CC (330 g). Elution was started with n-hexane followed by increasing polarity in gradient manner between n-hexane and EtOAc. Fractions of 200 ml each were collected and monitored using TLC and 10% v/v H2SO4 in MeOH as spraying reagent; similar fractions were pooled together where three groups (I-III) were obtained. Group I, fraction eluted with n-hexane:EtOAc (95:5), was further subjected to silica gel CC using mixture of n-hexane:EtOAc in gradient manner to give two sub fractions (I-1, I-2), compound (1) (25 mg) was precipitated from the latter one (I-2). Group II, fraction eluted with n-hexane:EtOAc (90:10), was further subjected to silica gel CC using mixture of n-hexane:EtOAc in gradient manner to give  compound (2) (20 mg) after crystallization with methanol and and compound (3) (40 mg).

On the other hand, a part of the chloroformic fraction (8 g) was chromatographed on silica gel CC (270 g). Elution was started with n-hexane followed by mixture of n-hexane:EtOAc in gradient manner, fractions of 200 ml were collected, and three groups of fractions (I-III) were obtained. Group II, fraction eluted with n-hexane:EtOAc (20:80), was further chromatographed over silica gel CC using CHCl3:MeOH in gradient manner to give compound (4) (30 mg). Group III, fraction eluted with n-hexane:EtOAc (5:95) precipitated compound (5) (60 mg).        Finally, a part of the EtOAc fraction (12 g) was subjected to silica gel CC (360 g), running with CHCl3:MeOH in gradient manner, where four groups of fractions (I-IV) were obtained. Group II, fraction eluted with CHCl3:MeOH (85:15), was further subjected to silica gel CC using the same solvent mixture to yield two sub fractions (1,2); compound (6) (12 mg) was precipitated from sub-fr. 2 (80:20). Group III, fraction eluted with CHCl3:MeOH (80:20), was subjected to Sephadex gel LH-20 column using CHCl3:MeOH (1:1), followed by silica gel CC using EtOAc: MeOH in gradient manner to yield three sub fractions (1-3); subfr. 3 (84:16) gave compound (7) (14 mg). Group IV, fraction eluted with CHCl3:MeOH (70:30), was subjected to Sephadex LH-20 column using CHCl3:MeOH (1:1), followed by silica gel CC using EtOAc: MeOH to give compound (8) (40 mg).

2.4 Chemicals for Biological Assays

Indomethacin was obtained as Liometacin from El-Nile Company for Pharmaceutical and Chemical Industries, Cairo, Egypt. Acetyl salicylic acid was obtained as (Aspirin 500, Bayer, Egypt). Metformin was obtained as (Cidophage 500, Chemical Industries Development Company "CID", Egypt). Alloxan monohydrate was obtained from Sigma Company, U.S.A, while other chemicals were obtained from El-Nasr Company for Pharmaceutical Chemicals "ADWIC", Egypt.

2.5 Animals

Adult Albino rats (each 150–200 g) of either sex were bred and housed under standardized environmental conditions in the pre-clinical animal house, Pharmacology Department, Faculty of Medicine, Assiut University, Assiut. The animals were fed with standard diet and free access to water and were kept for one week to acclimatize to the environmental conditions. The animals were handled only at the time of experiments and during cage cleaning. All conditions were made to minimize animal suffering.

2.6 Toxicity Study

The acute toxicity study of the methanolic extract of Russelia equisetiformis Sch. & Cham. was performed by measuring the lethal dose for 50% of the laboratory animals (LD50 method)17. The rats were kept fasting overnight with free access to water. They were divided into seven different groups (six rats per group) and administered different doses of the extract (0.5, 1, 2, 4, 8 and 16 g/Kg, p.o.) respectively suspended in the vehicle (0.5% carboxymethyl cellulose aqueous solution "CMC") beside control group which received an equivalent dose of the vehicle, orally. All of the groups’ animals were then allowed free access to food and water and observed a period of 48 hrs for signs of acute toxicity. The number of death within this period was recorded.

2.7 Anti-inflammatory Activity (yeast-induced Paw Oedema Method)

Different fractions of R. equisetiformis were evaluated for their anti-inflammatory activity18. Rats were randomly divided into seven groups (six rats per group). Group1 (negative control) was administered the vehicle (0.5 % CMC solution) orally, Group 2 was administered indomethacin (8 mg/kg) as the reference drug in vehicle orally, while groups 3-7 were administered 300 mg/kg of fractions A-E, respectively, suspended in the vehicle orally. After 30 minutes, inflammation was induced by subcutaneous injection of an equal volume of 20% w/v yeast aqueous suspension in the right hind paw of each rat under the sub-plantar region. The increase in linear paw circumference measured by a vernier caliber (at 0, 1, 2, 3, 4 and 5hrsafter yeast injection) was taken as a measure of oedema.

(%Inhibition) = [(Vo-Vt)/ Vo] . 100,

Where, Vo– the average paw thickness of the control group,

Vt– the average paw thickness of the treated group. 

2.8 Antipyretic Activity

            For screening of the antipyretic activity, pyrexia was induced by subcutaneous injection of 20% w/v yeast aqueous suspension in the back, below the nape of the neck. After 18 hours, the same grouping of animals in the anti-inflammatory study and their respective treatment were followed except group 2 (positive control) which received acetyl salicylic acid 100 mg/kg as the reference drug in vehicle orally. The rectal temperature before and after treatment, which was recorded with the help of a digital thermometer at every hour up to four hours, was compared with control19.

2.9 Antihyperglycemic Activity (Alloxan-induced hyperglycemia method)

For evaluation of antihyperglycemic activity of the different fractions of R. equisetiformis, the rats were subjected to a 12-hour fast. Diabetes was induced by intraperitoneal injection of alloxan monohydrate at a dose of 150 mg/kg. Diabetic rats were divided into seven groups (six rats each). Group 1 (negative control) was given the vehicle, group 2 (positive control) was given metformin 500 mg/kg as the reference drug in vehicle, while the other five groups received the tested fractions at a dose level of 300 mg/kg of fractions A-E, respectively, suspended in the vehicle. All treatments were given orally. Blood glucose level was measured by a glucometer at every hour up to five hours and compared with the control20.

2.10 Statistical analysis

The results were analyzed for statistical significance by one-way analysis of variance (ANOVA) test using the statistical package of the social science (SPSS) program. The results are presented as the mean ± S.E. (standard error). Group means were compared by a one-way analysis of variance and Duncan’s multiple range tests. Statistical differences were considered significant at P<0.05 and very significant at P<0.01.

Compound (1) (n-pentatriacontane) was obtained as a white waxy powder, m.p. 74-76 °C, Rf =0.37 (system II). 1H-NMR spectral data (CDCl3, 600 MHz) dH: 0.86 (6H, t, H3-1, H3-35), 1.23 (64H, brs, 32×H2), 1.59 (2H, brs, H2). 13C-NMR spectral data (CDCl3, 150 MHz) dC: 14.2 (C-d), 22.7 (C-?), 29.4 (C-a), 29.7 (27×C), 32.0 (C-ß). EI-MS showed a molecular ion peak at m/z 494 [M]+, corresponding to the molecular formula (C35H72).

                Compound (2) (a mixture of ß-Sitosterolandstigmasterol) was obtained as colourless needles, m.p. 159-160 °C, Rf =0.44 (system I).The mixture was identified by comparing its NMR data with those previously reported21.


                Compound (3) (lupeol) was obtained as white crystals, m.p. 215-216 °C, Rf =0.51 (system I). 1H-NMR spectral data (C5D5N, 400 MHz) dH: 0.83 (3H, s, H-28), 0.89 (3H, s, H-27), 0.98 (3H, s, H-23), 1.03(3H, s, H-25), 1.05 (3H, s, H-26), 1.23 (3H, s, H-24), 1.74 (3H, s, H-30), 2.48 (H, m, H-19), 3.46 (H, m, H-3), 4.70 (H, s, H-29a), 4.90 (H, s, H-29b). 13C-NMR spectral data (C5D5N, 100 MHz) dC: 14.5 (C-27), 16.0(C-24), 16.1(C-26), 16.2(C-25), 18.0 (C-28), 18.6 (C-6), 19.5 (C-30),20.9(C-11), 25.4 (C-12), 27.6 (C-15), 28.1(C-2), 28.5 (C-23), 30.0 (C-21), 34.5 (C-7), 35.6 (C-16), 37.3 (C-10), 38.2 (C-13), 39.1 (C-1), 39.3 (C-4), 40.0 (C-22), 40.9 (C-8), 43.0 (C-14), 43.0 (C-17), 48.1 (C-19), 48.4 (C-18), 50.6 (C-9), 55.7 (C-5), 77.9 (C-3), 109.7 (C-29), 150.8 (C-20).


                Compound (4) (a mixture of ß-sitosterol- and stigmasterol-1-O-ß-D-glucopyranoside) was obtained as a white amorphous powder, m.p. 265 °C, Rf0.23 (system III). The mixture was identified by comparing its NMR data with those previously reported22.

Compound (5) (4-allyl-2,6-dimethoxyphenol-1-O-ß-D-glucopyranoside) was obtained as colourless needles (methanol), m.p. 159-161 °C, Rf 0.54 (system IV). 1H-NMR spectral data (CD3OD, 600 MHz) dH: 3.33 (2H, d, J=6.6, H-a), 3.66 (H, dd, J=12, 5.4, H-6'a), 3.77 (H, dd, J=12, 2.4, H-6'ß), 3.81 (2×H3, s, OCH3), 4.80 (H, d, J=7.8, H-1'), 5.10 (2H, m, H-?), 5.90 (H, m, H-ß), 6.52 (2×H, s, H-3, H-5). 13C-NMR spectral data (CD3OD, 150 MHz) dC: 41.4 (C-a), 56.9 (2×OCH3), 62.6 (C-6'), 71.3 (C-4'), 75.7 (C-2'), 77.8 (C-5'), 78.3 (C-3'), 105.5 (C-1'), 107.4 (C-3, C-5), 116.2 (C-?), 134.6 (C-4), 138.4 (C-1), 138.7 (C-ß), 154.2 (C-2, C-6).

 

 

 

                Compound (6) (vitexin) was obtained as a yellow powder from methanol, m.p. 262-264 °C, Rf 0.38 (system V). 1H-NMR spectral data (DMSO, 600 MHz) dH: 4.67 (H, d, J=10.2, H-1?), 6.25 (H, s, H-6), 6.76 (H, s, H-3), 6.88 (2×H, d, J=8.4, H-3', H-5'), 8.01 (2×H, d, J=8.4, H-2', H-6'), 13.15 (H, brs, OH). 13C-NMR spectral data (DMSO, 150 MHz) dC: 61.4 (C-6?), 70.6 (C-4?), 70.9 (C-2?), 73.5 (C-1?), 78.7 (C-3?), 81.9 (C-5?), 98.3 (C-6), 102.5 (C-3), 104.1 (C-10), 104.7 (C-8), 115.9 (C-3', C-5'), 121.7 (C-1'), 129.1 (C-2', C-6'), 156.1 (C-9), 160.5 (C-5), 161.2 (C-4'), 162.8 (C-7), 164.1 (C-2), 182.2 (C-4).


                Compound (7) (acteoside) was abtained as a yellowish amorphous powder, m.p. 135-136 °C, Rf 0.56 (system VI). 1H-NMR spectral data (CD3OD, 600 MHz) dH: 1.08 (3H, d, J=6.2, H-6?), 2.79 (2H, m, H-7), 3.28 (H, m, H-4?), 3.38 (H, t, H-2?), 3.52-3.63 (5H, m, overlapped, H-5?, H-6?, H-3?, H-5?), 3.71 (H, m, H-8a), 3.80 (H, t, J=9.2, H-3?), 3.91 (H, m, H-2?), 4.05 (H, m, H-8ß), 4.37 (H, d, J=7.9, H-1?), 4.92 (H, t, J=9.2, H-4?), 5.17 (H, brs, H-1?), 6.27 (H, d, J = 15.9, H-8'), 6.55 (H, dd, J=2, 8, H-6), 6.68 (H, d, J=8, H-5), 6.70 (H, d, J=2, H-2), 6.76 (H, d, J=8, H-5'), 6.95 (H, dd, J=2, 8, H-6'), 7.05 (H, d, J=2, H-2'), 7.58 (H, d, J=15.9, H-7'). 13C-NMR spectral data (CD3OD, 150 MHz) dC:18.5 (C-6?), 36.6(C-7), 62.4 (C-6?), 70.4 (C-5?), 70.6 (C-4?), 72.1 (C-3?), 72.3 (C-8), 72.4 (C-2?),73.8 (C-4?), 76.1 (C-5?), 76.2 (C-2?), 81.7 (C-3?), 103.1 (C-1?), 104.2 (C-1?),114.7 (C-8'), 115.2 (C-2'), 116.3 (C-5), 116.5 (C-5'),117.11 (C-2), 121.2 (C-6), 123.2 (C-6'), 127.6 (C-1'), 131.4 (C-1), 144.7 (C-4), 146.2 (C-3), 146.9 (C-3'), 148.0 (C-7'), 149.8 (C-4'), 168.3 (C-9').


                Compound (8) (isoacteoside) was obtained as a yellowish amorphous powder, m.p. 134-135 °C, Rf0.38 ( system VI ).1H-NMR spectral data (CD3OD, 600 MHz) dH: 1.24 (3H, d, J=6.2, H-6?), 2.78 (2H, m, H-7), 3.28 (H, m, H-4?), 3.38 (H, t, H-2?), 3.40 (H, t, J=9.2, H-4?), 3.52-3.63 (5H, m, overlapped, H-5?, H-6?, H-3?, H-5?), 3.71 (H, m, H-8a), 3.80 (H, t, J=9.2, H-3?), 3.92 (H, m, H-2?), 4.05 (H, m, H-8ß), 4.33 (H, d, J=7.9, H-1?), 5.18 (H, brs, H-1?), 6.29 (H, d, J = 15.9, H-8'), 6.55 (H, dd, J=2, 8, H-6), 6.68 (H, d, J=8, H-5), 6.70 (H, d, J=2, H-2), 6.75 (H, d, J=8, H-5'), 6.88 (H, dd, J=2, 8, H-6'), 7.03 (H, d, J=2, H-2'), 7.56 (H, d, J=15.9, H-7'). 13C-NMR spectral data (CD3OD, 150 MHz) dC: 17.9 (C-6?), 36.7(C-7), 64.4 (C-6?), 70.0(C-5?), 70.3 (C-4?), 72.3(C-3?), 72.4 (C-8), 72.5(C-2?),74.0 (C-4?), 75.4 (C-5?), 75.7(C-2?), 84.0(C-3?), 102.7(C-1?), 104.4(C-1?), 114.8 (C-8'), 115.1(C-2'), 116.3 (C-5), 116.5 (C-5'),117.11  (C-2), 121.2 (C-6), 123.2 (C-6'), 127.6 (C-1'), 131.3(C-1), 144.7 (C-4), 146.2 (C-3), 146.8(C-3'), 147.3(C-7'), 149.7(C-4'), 169.1(C-9').

3. RESULTS

From the aerial parts of R. equisetiformis, eight compounds were isolated using different chromatographic techniques and identified by different physical, chemical, and spectroscopic methods in addition to comparison with reported data.

Compound (1) was obtained as a white waxy powder from the n-hexane fraction and was identified with the aid of 1D (1H and13C) NMR data and mass spectroscopy as n-pentatriacontane23. Compounds (2-4) were obtained from the n-hexane and chloroform fractions and gave positive results with Salkowski’s and Liebermann-Burchard’s tests indicating their triterpenoidal and/or steroidal nature, andfrom their NMR data together with co-chromatography, the compounds were identified as a mixture of ß-sitosterol and stigmasterol(2)21, lupeol (3)24, a mixture of ß-sitosterol- and stigmasterol-1-O-ß-D-glucopyranosides (4)22. Compound (5) was obtained as colourless needles from the chloroform fraction and was identified as 4-allyl-2,6-dimethoxyphenol-1-O-ß-D-glucopyranoside25. From the ethyl acetate fraction, compounds (6-8) were isolated and identified as vitexin (6)26, acteoside (7) and isoacteoside (8)27-28.

Different biological studies were carried out to evaluate the acute toxicity, anti-inflammatory, antipyretic as well as antihyperglycemic activity of the methanolic extract and all the fractions. For acute toxicity study, the methanolic extract was evaluated and the LD50 value was found to be 8 g/kg.

 

Thickness of the paw (mm)

Dose

mg/kg

 

Group

5h

4h

3h

2h

1h

0h

7.62 ± 0.10

7.62 ± 0.10

7.64 ± 0.10

7.48 ± 0.16

7.28 ± 0.12

3.42 ± 0.19

-

Control (negative)

3.4 ± 0.10**

3.32 ± 0.13**

3.76 ± 0.11**

3.9 ± 0.10**

5.78 ± 0.13**

3.24 ± 0.11

8

Indomethacin

6.76 ± 0.11**23

6.34 ± 0.14**

5.56 ± 0.17**

6.14 ± 0.10**

7.16 ± 0.10

3.36 ±0.10

300

Total

extract

5.28 ± 0.12**

4.76 ± 0.11**

5.14 ± 0.10**

5.88 ± 0.10**

5.62 ± 0.10**

3.12 ± 0.10

300

n-Hexane fraction

3.5 ± 0.03**

4.02 ± 0.02**

4.02 ± 0.02**

4.64 ± 0.10**

5.12 ± 0.10**

3.26 ± 0.11

300

Chloroform fraction

4.88 ± 0.10**

4.86 ± 0.10**

4.76 ± 0.11**

5.62 ± 0.10**

6.12 ± 0.10**

3.28 ± 0.16

300

Ethyl acetate fraction

6.86 ± 0.10**

6.3 ± 0.34**

5.72 ± 0.16**

6.16 ± 0.10**

6.8 ± 0.02*

3.18 ± 0.10

300

Aqueous fraction

*P<0.05, **P<0.01

Percentage inhibition (%)

Dose

mg/kg

 

Group

5h

4h

3h

2h

1h

-

-

-

-

-

-

Control (negative)

55.38

56.43

50.79

47.86

20.60

8

Indomethacin

11.29

16.80

27.23

17.90

1.65

300

Total extract

30.71

37.53

32.59

21.39

22.80

300

n-Hexane fraction

54.07

47.24

47.38

37.97

29.67

300

Chloroform fraction

35.96

36.22

37.70

24.87

15.93

300

Ethyl acetate fraction

9.97

17.32

25.13

17.65

6.59

300

Aqueous fraction

For anti-inflammatory activity, different fractions of R. equisetiformis were evaluated using yeast-induced paw edema method in rats. The chloroform fraction exhibited a significant anti-inflammatory activity at a dose (300 mg/kg) which significantly reduced the yeast-induced hind paw edema in rats compared with indomethacin at a dose 8 mg/kg. The n-hexane and ethyl acetate fractions exhibited a moderate activity while both the total and aqueous fractions were weakly active (Table 1 & 2).

 

For antipyretic activity, the ethyl acetate fraction (300 mg/kg) showed a high and significant activity after 1h from pyrexia induction using yeast and up to 4h compared with acetyl salicylic acid (100 mg/kg) as a positive control (Table 3). The other fractions exhibited a moderate and significant activity after 2h and up to the end of the experiment (4h).

 

 

Group

Dose

mg/kg

Average rectal temperature (°C)

Before yeast injection

 

Pre-drug

 

1h

2h

3h

4h

Control (negative)

-

36.92 ± 0.03

39.15 ± 0.09

39.17 ± 0.12

39.82 ± 0.03

39.83 ± 0.02

39.83 ± 0.02

Acetylsalicylic acid

100

36.92 ± 0.04

38.87 ± 0.04*

37.48 ± 0.09**

36.50 ± 0.02**

35.42 ± 0.03**

35.52 ± 0.03**

Total extract

300

36.90 ± 0.04

38.88 ± 0.05*

38.85 ± 0.05*

36.78 ± 0.03**

36.40 ± 0.19**

36.10 ± 0.03**

n-Hexane fraction

300

36.92 ± 0.04

38.90 ± 0.04*

38.9 ± 0.04

37.93 ± 0.08**

36.17 ± 0.02**

36.10 ± 0.03**

Chloroform fraction

300

36.88 ± 0.03

39.03 ± 0.04

39.03 ± 0.04

37.05 ± 0.03**

35.90 ± 0.04**

35.90 ± 0.04**

Ethyl acetate fraction

300

36.92 ± 0.04

38.60 ± 0.11**

38.60 ± 0.11**

36.90 ± 0.04**

36.00 ± 0.05**

35.63 ± 0.04**

Aqueous fraction

300

36.90 ± 0.04

38.93 ± 0.04

38.93 ± 0.04

37.38 ± 0.28**

35.93 ± 0.03**

35.90 ± 0.04**

 

*P<0.05, **P<0.01

Group

Dose

mg/kg

Blood glucose level (mg./dl)

0h

1h

2h

3h

4h

5h

Control (negative)

120

476.17 ± 4.93

470.83 ± 9.12

471.5 ± 11.56

465 ± 15.17

465 ± 22.84

481 ± 14.66

Metformin

500

447.83 ± 6.43

384 ± 11.67**

312.17 ±13.76**

231.5 ± 4.63**

143.33 ± 2.95**

124.5 ± 2.98**

Total extract

300

461 ± 17.03

395 ± 2.22**

325 ± 7.65**

255.33 ± 8.04**

167.5 ± 2.45**

191.33 ± 5.74**

n-Hexane fraction

300

483.17 ± 6.26

470.17 ± 7.31

476.5 ± 5.81

458.17 ± 14.39

472 ± 11.60

494.5 ± 15.60

Chloroform fraction

300

470.67 ± 17.37

392.33 ± 4.90**

303 ± 18.05**

239.17 ±10.55**

173.5 ± 5.81**

196 ± 9.68**

Ethyl acetate fraction

300

480.17 ± 7.02

381.5 ± 7.75**

279.17 ±4.23**

204 ± 10.53**

150.67 ± 4.99**

183.33 ± 4.37**

Aqueous fraction

300

457.67 ± 15.34

442.83 ± 8.39*

430 ± 14.68

444 ± 15.66

482.33 ± 7.97

484.33 ± 7.89

 

*P<0.05, **P<0.01

For the antihyperglycemic activity, the total extract, chloroform, and ethyl acetate fractions (300 mg/kg) exhibited a high and significant activity after 1h from hyperglycemia induction using alloxan and up to 5h, compared with metformin (500 mg/kg) as a positive control (Table 4). Both n-hexane and aqueous fractions were inactive.

 

4. DISCUSSION

Eight known compounds including sterols, triterpenoids, flavonoids and phenethyl caffeoyl glycosides were isolated from the methnolic extract of the aerial parts of R. equisetiformis. The isolation of compounds (1-4) is considered as the first report for the isolation of such compounds from R. equisetiformis which could be helpful and can contribute in the chemotaxonomic analysis of this genus which has no previous studies. These compounds can also contribute in explanation of many of the biological activities exhibited by R. equisetiformis. The acute toxicity study showed that the methanolic extract with its high LD50 value showed a wide safety margin which could explain the wide use of R. equisetiformis in folkloric medicine. The different biological assays for the total extract and the different fractions showed that the chloroform fraction, together with the n-hexane and ethyl acetate ones were the most active against inflammation. Furthermore, ethyl acetate fraction showed a significant antipyretic activity could be attributed to its high content of flavonoids29.

                Finally, the antihyperglycemic activity showed that the total extract, chloroform and ethyl acetate fractions were significantly active against hyperglycemia, especially the latter which had the best effect reducing BGL up to 5h, and in comparison with metformin, having a better effect after 2 and 3h. From the overall results of the current study, it can conclude that both the ethyl acetate and chloroform fractions possess promising anti-inflammatory, antipyretic and antihyperglycemic activities.

 

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Editor in Chief
Prof. Dr. Cornelia M. Keck (Philipps-Universität Marburg)
Marburg, Germany

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Welcome to the research group of Prof. Dr. Cornelia M. Keck in Marburg. Cornelia M. Keck is a pharmacist and obtained her PhD in 2006 from the Freie Universität (FU) in Berlin. In 2009 she was appointed as Adjunct Professor for Pharmaceutical and Nutritional Nanotechnology at the University Putra Malaysia (UPM) and in 2011 she obtained her Venia legendi (Habilitation) at the Freie Universität Berlin and was appointed as a Professor for Pharmacology and Pharmaceutics at the University of Applied Sciences Kaiserslautern. Since 2016 she is Professor of Pharmaceutics and Biopharmaceutics at the Philipps-Universität Marburg. Her field of research is the development and characterization of innovative nanocarriers for improved delivery of poorly soluble actives for healthcare and cosmetics. Prof. Keck is executive board member of the German Association of Nanotechnology (Deutscher Verband Nanotechnologie), Vize-chairman of the unit „Dermocosmetics“ at the German Society of Dermopharmacy, active member in many pharmaceutical societies and member of the BfR Committee for Cosmetics at the Federal Institute for Risk Assessment (BfR).

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doi: http://dx.doi.org/10.21065/19204159
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