Chemical and biological investigation of some Clerodendrum species cultivated in Egypt

Abstract

Context: Phytochemical investigation of Clerodendrum chinense (Osbeck) Mabberley (Lamiaceae) cultivated in Egypt and evaluation for anti-inflammatory, analgesic, and antipyretic effects of the methanol and chloroform extracts of Clerodendrum chinense, Clerodendrum indicum (L.) Kuntze, Clerodendrum glabrum E. Meyer.

Objective: The objective of this study was to investigate the anti-inflammatory, analgesic, and antipyretic effects of the methanol and chloroform extracts of Clerodendrum species under investigation.

Materials and methods: Air-dried powdered leaves of C. chinense were extracted with MeOH 80%. This extract was fractionated with successive portions of chloroform, ethyl acetate and n-butanol. By further fractionation through silica gel, polyamide and reversed phase column chromatography several compounds were isolated which were elucidated by nuclear magnetic resonance (NMR) and mass spectroscopy. For biological study, the powdered leaves of C. chinense, C. indicum and C. glabrum were extracted by chloroform and then extracted with methanol. The acute anti-inflammatory effect of tested extracts of the leaves of Clerodendrum species under investigation was estimated by carrageenan-induced rat paw edema. Antipyretic effect was evaluated and compared with that of paracetamol as standard using the yeast-induced hyperthermia method on female albino rats. Analgesic effect was evaluated and compared with that of Novalgin (metamizol sodium) as standard using an electric current anxious stimulus.

Results: Verbascoside, isoverbascoside, decaffeoylverbascoside, hispidulin, lupeol and icariside B5 were isolated from the leaves of C. chinense for the first time. Cornoside and rengyolone were also isolated. The methanol extract of the leaves of C. chinense and verbascoside showed significant analgesic, anti-inflammatory and antipyretic effects.

Conclusion: The present study provided a scientific validation of the traditional claims suggested for C. chinense.

Keywords::

• Analgesic
• anti-inflammatory
• antipyretic
• Clerodendrum chinense
• Clerodendrum glabrum
• Clerodendrum indicum
• verbascoside

Introduction

The genus Clerodendrum (Lamiaceae) is widely distributed and has been used for ages in Indian and Chinese traditional medicine (Rajasekaran & Ponnusamy, 2006). In Nepal, the fresh juice of C. indicum (L.) Kuntze is used for the treatment of fever (Taylor et al., 1996). In Thailand, C. chinense (Osbeck) Mabberley is used for its anti-pyretic and anti-inflammatory effects (Kanchanapoom et al., 2005). Unfortunately, there is no scientific study to support these traditional claims. Therefore, we conducted this research work to investigate the anti-inflammatory, antipyretic, analgesic effects of C. chinense, C. indicum and C. glabrum E. Meyer. Phytochemical investigation of the leaves of C. chinense was carried out. Three phenylpropanoid glycosides (verbascoside, isoverbascoside and decaffeoylverbascoside), one flavonoid (hispidulin), two cyclohexylethanoids (cornoside and rengyolone) and icariside B5 were isolated. The structures of isolated compounds were elucidated on the basis of spectroscopic data.

Material and methods

General

NMR spectra were recorded on a Bruker DRX-400 instrument operating at 400 MHz for ¹H-NMR at 30°C in CD₃OD (99.5% Deutrated (D)), CDCl₃ (99.8% D) or DMSO-d₆ (99.5% D). Chemical shifts were recorded in δ (ppm) relative to TMS (0 ppm). Mass spectra were recorded on a Bruker Esquire 3000 Plus Ion trap Mass Spectrometer with ESI and Esquire Control software. UV-visible spectra were recorded on a Shimadzu UV 240 spectrophotometer.

Chromatographic materials included silica gel 60 for CC (Davisil), reversed phase Lichroprep^® RP-18 (40-63 µm) for CC (Merck), Polyamide CC 6 (0.05-0.16 mm) for CC (Macherey-Nagel), and TLC plates (silica gel 60 F₂₅₄-Merck). p-Anisaldehyde-sulfuric acid spray reagent (Stahl, 1969), sodium methoxide solution, aluminum chloride solution, hydrochloric acid, sodium acetate powder and boric acid powder were used as reagents for UV-spectroscopic analysis of flavonoids (Mabry et al., 1970). All of the solvents used were of HPLC grade.

Collection of plant material

The leaves of C. chinense were collected from plants cultivated in Orman Botanical Garden, Giza, Egypt. The leaves of C. indicum were collected from plants cultivated in Giza Zoo, Giza, Egypt. The leaves of C. glabrum were collected from plants cultivated in Al-Zohrya Botanical Garden, Giza, Egypt. Collection of the plant materials was carried out in September 2006. The plants were identified by Mohamed Elgebaly, Department of Chemotaxonomy, National Research Centre, Cairo, Egypt. Voucher specimens were prepared and deposited at the Department of Pharmacognosy, Faculty of Pharmacy, Beni-Sueif University, Egypt.

Preparation of extracts for biological study

The leaves of the plants under investigation were separately air-dried, powdered and kept in amber-colored, well-closed containers at low temperature. The following procedure was followed for each plant. The leaves (100 g) were extracted with chloroform then the residual plant materials were allowed to dry, then extracted with methanol. The yields from chloroform and methanol extraction were 4.4, 4.8, 3.3 g and 10, 8.8, 11 g for C. chinense, C. indicum, and C. glabrum, respectively.

Extraction and isolation

Air-dried powdered leaves of C. chinense (900 g) were extracted with aqueous MeOH 80% (2 × 2000 mL, 72 h each). The solvent was evaporated under reduced pressure. The extract was dissolved in water and left overnight in a refrigerator, then filtered. The filtrate was extracted with successive portions of chloroform, ethyl acetate and n-butanol. The organic phases were evaporated to dryness under reduced pressure. The chloroform, ethyl acetate and n-butanol extracts yielded a residue of 1.5, 9 and 24 g, respectively. About 5 g of the dried ethyl acetate extract was applied on Silica gel column using n-hexane:ethyl acetate (50:50), gradually increasing the polarity untill 100% ethyl acetate; then the polarity was further increased using methanol (100 mL fractions). Forty fractions were collected and examined by TLC using ethyl acetate: hexane 7.5:2.5 v/v and/or ethyl acetate:methanol:water 9:1:1 v/v as the mobile phase. Fractions 5–6 (216 mg) were subjected to silica gel column to afford compound f1 (20 mg yellow crystals). Fractions 19–23 (3 g) were combined and subjected to polyamide column chromatography using a water/methanol gradient; 76 fractions were collected. The fractions were examined by TLC using the solvent system ethyl acetate:formic acid:acetic acid:water (30:1.2:0.8:8). Fractions 14–17 (330 mg) were combined and subjected to RP C18 column chromatography using water and increasing amounts of acetonitrile (50 mL fractions) to afford compound f2 (100 mg orange powder). Fractions 18–22 (350 mg) were combined and subjected to RP C18 column chromatography using water and increasing amounts of acetonitrile (52 mL fractions) to afford compound f3 (10 mg greenish powder). The n-butanol fraction (15 g) was subjected to polyamide column chromatography using water and increasing amounts of methanol; 80 fractions were collected and examined by TLC using the solvent system ethyl acetate:formic acid:acetic acid:water (30:1.2:0.8:8) and vanillin/sulfuric acid as spray reagent. Fractions 12–23 (5 g) were combined and subjected to RP C18 column chromatography using water and acetonitrile; 180 fractions were collected. Fractions 90–98 (100 mg), fractions 159–161 (50 mg) and fractions 173–175 (25 mg) were separately combined and further fractionated on a RP C18 column using water and acetonitrile to afford b1 (8 mg colorless oil), compound b2 (10 mg orange powder) and compound b3 (8 mg colorless oil), respectively. About 450 g air-dried powdered leaves of C. chinense were extracted with chloroform. The solvent was evaporated to dryness under reduced pressure (10 g). The residue was subjected to silica gel column chromatography using n-hexane and increasing amounts of ethyl acetate (50 ml fractions); 50 fractions were collected and examined by TLC using ethyl acetate:n-hexane (6:4). Fraction 24 afforded compound d1 (5 mg white powder). Repeated silica gel column chromatography of combined fractions 33–40 (250 mg) afforded compound d2 (70 mg colorless oil).

Biological study

Animals

Adult male albino rats of Sprague-Dawley strain (130–150 g body weight) and adult female albino rats of Sprague-Dawley strain (100 g body weight) were obtained from the animal house, National Research Center, Giza, Egypt. All the animals were kept under the same hygienic conditions of 25° ± 1°C, 12 h light-dark cycle and 55% ± 5% humidity. The animals were given water and a standard laboratory diet consisting of vitamin mixture (1%), mineral mixture (4%), corn oil (10%), sucrose (20%), cellulose (0.2%), casein 95% pure (10.5%) and starch (54.3%) ad libitum. All of the methods used in the present study were approved by the ethics committee of National Research Center.

Anti-inflammatory activity

This activity was determined according to a reported procedure (Winter et al., 1962). Fifty-four male albino rats, weighing 130–150 g were divided into nine groups of six rats each. The first group received 1 mL of saline serving as control, the second group received 20 mg/ kg body weight indomethacin (Epico, Cairo, Egypt) (orally) serving as standard anti-inflammatory drug. The other groups received the test extracts of different Clerodendrum species under investigation using a dose of 100 mg/kg body weight (orally) and a dose of 25 mg/ kg body weight (orally) for the isolated verbascoside. After 1 h all animals received a subplantar injection of 0.1 mL of 1% carrageenan solution (Sigma, USA) in saline in the right hind paw, and 0.1 mL saline in the left hind paw. Four hours after drug administration the rats were sacrificed; both hind paws excised and weighed separately. The percentage of edema and inhibition was calculated.

Analgesic activity

This activity was evaluated according to a reported procedure (Charlier et al., 1961). Male albino rats (54, weighing 130–150 g) were divided into nine groups of six rats each. The first group received 1 mL saline serving as control, the second group received 50 mg/kg body weight Novalgin (metamizol sodium) (Hoechst Orient, Cairo, Egypt) (orally) serving as standard analgesic drug. The other groups received extracts of different Clerodendrum species under investigation using a dose of 100 mg/ kg body weight (orally), and a dose of 25 mg/kg body weight (orally) for the isolated verbascoside. Electrical stimulation was applied to the rats’ tail by means of 515 Master Shocker (Lafayette, USA), using AC (50 Hz) for 0.2 s. The minimum voltage required for the animal to emit a cry was recorded before treatment at zero time (V₀), after 1 h, and after 2 h intervals for the treated group (V_t), in order to calculate percentage change.

Antipyretic activity

Antipyretic activity was tested using the yeast-induced hyperthermia method according to a reported procedure (Bush & Alexander, 1960). The results were compared with that of paracetamol as standard drug. The initial vaginal temperature of female albino rats of average weight 100 g were recorded with a digital thermometer (SK-1250MC, Sato Keiryoki, Tokyo, Japan). The probe was attached to a digital display and was inserted 1 cm into the vagina. Pyrexia was induced by subcutaneous injection of 1 mg/100 g body weight of 44% (w/v) brewer’s yeast suspension in physiological saline into the animal’s dorsum region. When the temperature was at a peak, 18 h after yeast injection, only rats which developed satisfactory pyrexia (1°C or more increase in vaginal temperature) were divided into nine groups of six rats each. At this point the vaginal temperature was recorded for all groups to serve as the base line of elevated body temperature, to which the antipyretic effect will be compared. The first group received 1 mL of saline serving as control, the second group received 20 mg/kg body weight paracetamol (Misr, Cairo, Egypt) as standard antipyretic drug. The other groups received the test extracts of different species under investigation using a dose of 100 mg/kg body weight (orally), and a dose of 25 mg/kg body weight (orally) for the isolated verbascoside. Vaginal temperatures were recorded immediately before test extracts, paracetamol and saline administration and at 19 and 20 h after yeast injection, and the percentage change was calculated.

Statistics

The data are the mean of triplicate measurements. The results are expressed as mean ± SE, (n = 6). Statistical significance was determined by Student’s t-test with P <0.01 considered significant (Snedecor & Cochran, 1971).

Results and discussion

Phytochemical study

Compounds f1-f3 (Figure 1) were identified as verbascoside and isoverbascoside by comparing their obtained physicochemical and spectral data with published data (Cooper et al., 1980; Andary et al., 1982; Miyase et al., 1982, 1991; Calis et al., 1984; Grande et al., 1985; Sasaki, et al. 1989; Liu et al., 1992; Hazekamp et al., 2001; Kim et al., 2001; Kanchanapoom et al., 2002). Compounds b1-b3 (Figure 1) were identified as cornoside, decaffeoylverbascoside and icariside B₅ (Jensen et al., 1973; Aasen et al., 1974; Endo & Hikino, 1984; Miyase et al., 1988; Kuwajima et al., 1993; Hase et al., 1995; Kanchanapoom et al., 2002). Compounds d1 and d2 (Figure 1) were identified as lupeol and rengyolone (Endo & Hikino, 1984; Nishino, et al., 1988; Bianco et al., 1993; Hase et al., 1995; Tian et al., 1997; Gauthier et al., 2006; Ahiahonu & Goodenowe, 2007; Siddiqui et al., 2007). Except for cornoside and rengyolone, all other compounds were isolated from C. chinense for the first time.

Figure 1.  The isolated compounds of C. chinense.

Biological study

Anti-inflammatory activity

It is well known that the carrageenan paw edema test produces an acute inflammation that results from the sequential action of several mediators through two phases (Vinegar et al., 1961). The early phase (1.5 h) is attributed to the release of histamine and serotonin (Crunkhon & Meacock, 1961), while the delayed phase (4 h) of inflammatory response has been linked to release of cyclooxygenase and prostaglandins (Panthong et al., 2004). Since the second phase (4 h) of inflammatory edema induced by carrageenan is sensitive to most clinically effective anti-inflammatory drugs, this assay is useful for studying the anti-edematous effect of natural products (Calixto et al., 2004).

Results (Table 1) revealed that the rat groups that received the methanol extract of the leaves of C. chinense and C. indicum at an oral dose of 100 mg/g body weight and verbascoside at an oral dose of 25 mg/kg body weight showed highly significant anti-inflammatory activity (about 77–89% of the activity of indomethacin after 4 h). The rat groups that received the chloroform extract of the leaves of C. chinense and the methanol extract of leaves of C. glabrum at an oral dose of 100 mg/kg body weight showed significant anti-inflammatory activity (about 56–63% of the activity of indomethacin after 4 h). Chloroform extracts of the leaves of both C. indicum and C. glabrum showed less anti-inflammatory activity at an oral dose of 100 mg/kg body weight.

Table 1.  Anti-inflammatory activity of different extracts of different Clerodendrum species and verbascoside in male albino rats

Treatment	Dose(mg/kg bw)	% Edema
		Mean^c	% change ^b
Control (saline 1 mL)		62.4 ± 2.1	-
Indomethacin	20	22.3 ± 0.6^a	64.26
Methanol extract of leaves of C. chinense	100	28.3 ± 0.4^a	54.64
Methanol extract of leaves of C. indicum	100	31.2 ± 1.2^a	50
Methanol extract of leaves of C. glabrum	100	39.6 ± 1.4^a	36.53
Chloroform extract of leaves of C. chinense	100	37.1 ± 1.1^a	40.54
Chloroform extract of leaves of C. indicum	100	49.2 ± 0.9^a	21.15
Chloroform extract of leaves of C. glabrum	100	47.8 ± 1.6^a	23.39
Verbascoside	25	26.5 ± 0.3^a	57.53

^aSignificantly different from control: P <0.01.

^bPercentage change calculated with regard to the control.

^cValues are expressed as mean ± SEM (n = 6).

Our results demonstrated that methanol extracts of C. chinense and C. indicum have an acute anti-inflammatory effect. The effects of these extracts may correlate to the inhibition of the enzyme cyclooxygenase, leading to the inhibition of prostaglandin synthesis. Verbascoside, present in C. chinense and C. indicum, may be responsible for this activity.

Analgesic activity

A significant increase in voltage required for the animal to emit a cry indicated the potent analgesic activity of the methanol extract of leaves of C. chinense and purified verbascoside.

Results (Table 2) revealed that the rat groups that received the methanol extract of the leaves of C. chinense at an oral dose of 100 mg/kg body weight and verbascoside at an oral dose of 25 mg/kg body weight showed highly significant analgesic activity (about 74–89% of the effect of Novalgin after 1 h and about 61–73% of the Novalgin effect after 2 h). Other groups showed less analgesic activity at an oral dose of 100 mg/kg body weight.

Table 2.  Analgesic activity of different extracts of different Clerodendrum species and verbascoside in male albino rats.

Treatment	Dose (mg/kg bw)	Volts needed before treatment (zero time)	Volts needed after single oral dose
			1 h		2 h
			Mean^c	% change^b	Mean^c	% change^b
Control (saline 1 mL)		79.3 ± 2.1	78.9 ± 1.8	0.5	78.4 ± 1.6	1.1
Novalgin	50	77.6 ± 2.8	147.8 ± 5.3^a	90.46	183.4 ± 6.2^a	136.34
Methanol extract of leaves of C. chinense	100	75.9 ± 2.3	137.2 ± 6.3^a	80.76	151.6 ± 7.1^a	99.73
Methanol extract of leaves of C. indicum	100	78.1 ± 1.9	102.9 ± 4.2^a	31.75	126.8 ± 5.7^a	62.35
Methanol extract of leaves of C. glabrum	100	79.9 ± 1.7	121.2 ± 5.3^a	51.68	130.5 ± 4.8^a	63.32
Chloroform extract of leaves of C. chinense	100	74.2 ± 1.6	97.8 ± 3.9^a	31.8	104.3 ± 5.2^a	40.56
Chloroform extract of leaves of C. indicum	100	76.5 ± 2.5	81.2 ± 2.7^a	6.14	119.3 ± 4.1^a	55.94
Chloroform extract of leaves of C. glabrum	100	78.4 ± 1.2	92.6 ± 3.5^a	18.11	123.4 ± 3.5^a	57.39
Verbascoside	25	76.8 ± 2.4	128.3 ± 5.1^a	67.05	141.2 ± 6.4^a	83.8

^aSignificantly different from zero time: p <0.01.

^bPercentage change calculated with regard to zero time.

^cValues are expressed as mean ± SEM (n = 6).

Antipyretic activity

Results (Table 3) revealed that the rat groups that received methanol extract of the leaves of C. chinense at an oral dose of 100 mg/kg body weight and verbascoside at an oral dose of 25 mg/kg body weight showed highly significant antipyretic activity (about 58-73% of the activity of paracetamol after 1 h and about 67-81% of paracetamol activity after 2 h). The rat group that received methanol extract of the leaves of C. glabrum at an oral dose of 100 mg/kg body weight showed significant antipyretic activity (about 57% of the activity of paracetamol after 1 h and about 55 % of paracetamol activity after 2 h).

Table 3.  Antipyrectic activity of different extracts of different Clerodendrum species and verbascoside in female albino rats.

Treatment	Dose (mg/kg bw)	Induced rise in temperature(zero time)	Body temperature change
			1 h		2 h
			Mean^c	% change^b	Mean^c	% change^b
Control (saline 1 mL)		38.9 ± 0.1	39.1 ± 0.3	–	39.3 ± 0.4	–
Paracetamol	20	39.4 ± 0.5	37.5 ± 0.1^a	4.82	36.7 ± 0.1^a	6.85
Methanol extract of leaves of C. chinense	100	39.3 ± 0.4	37.9 ± 0.1^a	3.56	37.1 ± 0.1^a	5.59
Methanol extract of leaves of C. indicum	100	38.7 ± 0.2	38.1 ± 0.2^a	1.55	37.7 ± 0.1^a	2.58
Methanol extract of leaves of C. glabrum	100	39.6 ± 0.5	38.5 ± 0.2^a	2.77	38.1 ± 0.2^a	3.78
Chloroform extract of leaves of C. chinense	100	39.3 ± 0.4	38.7 ± 0.3^a	1.52	38.5 ± 0.3^a	2.03
Chloroform extract of leaves of C. indicum	100	39.1 ± 0.3	38.8 ± 0.4	0.76	38.2 ± 0.2^a	2.3
Chloroform extract of leaves of C. glabrum	100	39.2 ± 0.2	38.9 ± 0.5	0.76	38.8 ± 0.4	1.02
Verbascoside	25	39.2 ± 0.3	38.1 ± 0.1^a	2.8	37.4 ± 0.1^a	4.59

^aSignificantly different from zero time: p <0.01 *P <0.01.

^bPercentage change calculated with regard to zero time (mean ± SE).

^cValues are expressed as mean ± SEM (n = 6).

The rat group that received methanol extract of the leaves of C. indicum at an oral dose of 100 mg/kg body weight showed less antipyretic activity. The rat groups that received chloroform extracts of the leaves of C. indicum or C. glabrum showed no significant antipyretic activity at an oral dose of 100 mg/kg body weight.

The present study demonstrated that C. chinense has significant analgesic, anti-inflammatory and antipyretic properties, justifying the traditional use of this plant in the treatment of inflammation.

Acknowledgment

Haytham M. Wahba would like to express his gratitude to Sheila M. Maregesi, Department of Pharmacognosy, School of Pharmacy, Muhimbili University, College of Health Sciences, Dar El Salaam, Tanzania for her kind and unlimited help.

Declaration of interest

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