Hypertension is a disease, which if left untreated affectsall important organs of human body. It is known as silentkiller as without showing significant symptoms, it may qui-etly lead to stroke, brain hemorrhage, cardiac disorders, renalfailure and vision loss. Hypertension has affected 10—15%of global population and killed a large number of human racein every region of world. Drugs used for its treatment on onehand are very expensive and beyond the reach of a commonman while on the other hand, side effects associated withthese drugs restricted the people to use them. Hence develop-ment of a cost effective antihypertensive drug with minimumor no side effects from commonly available indigenous medi-cinal plants is highly required. Keeping this in view as wellas blood pressure reducing property of Bombax ceiba presentwork describes the hypotensive evolution of its stem barkalongwith toxicology and histopathology of active fraction.

Bombax ceiba (syn. Bombax malabaricum DC) commonlyknown as silk cotton tree is an important medicinal plant.1,2)

Earlier investigations led to the identification of a sesquiter-pene lactone, a potent growth inhibitor in fungi3) and aflavonol glycoside possessing significant hypotensive and hy-poglycaemic activities.4) Both of these compounds were iso-lated from roots and leaves of B. ceiba respectively. This isthe first report of hypotensive evaluation of it’s stem barkwhich has resulted in the isolation of a novel benzopyrandimer, shamimicin, lupeol5) and fractions possessing hy-potensive activity. In addition to this, methanolic extracts ofdifferent parts of plant, including flowers, pulp, stem andstem bark have also been studied for their effect on mean ar-terial blood pressure (MABP) of rats.

MATERIALS AND METHODS

Instrumentation Ultraviolet spectra were recorded inMeOH on Hitachi-U-3200 and infrared spectra were mea-sured in CHCl3 on JASCO-A-302 spectrophotometers. Theelectron impact (EI) mass spectra were recorded on a Finni-gan MAT-112 instrument. Field desorption (FD-MS), fastatom bombardment (FAB-MS positive and negative) andexact mass measurements were carried out on a FinniganMAT-312 instrument. High-resolution mass spectra wererecorded on a JMS HX-110 spectrometer. The 1H- and 13C-NMR spectra were run in CDCl3 on a Bruker Aspect AM-

500 spectrometer operating at 500 MHz for 1H and 125 MHzfor 13C nuclei, with spectra referenced to residual solvent sig-nals. The chemical shifts are in ppm (d) and coupling con-stants (J) are in Hz. For flash column chromatography (FCC)and vacuum liquid chromatography (VLC) silica gel 9385(E. Merck) and silica gel 60 GF254 (Merck) were used, re-spectively. Purity of compounds was checked on silica gel 60GF254.

Plant Material Stem bark was collected in February2000 from Karachi University Campus. The plant was au-thenticated at the Department of Botany, University ofKarachi and a voucher specimen (No. 66854KU) was de-posited in the same department.

Isolation of Lupeol Fresh, uncrushed stem bark of B.

January 2003 Biol. Pharm. Bull. 26(1) 41—46 (2003) 41

∗ To whom correspondence should be addressed. e-mail: rs127pk@yahoo.com © 2003 Pharmaceutical Society of Japan

Hypotensive Activity and Toxicology of Constituents from Bombax ceibaStem Bark

Rubeena SALEEM,*,a Syed Iqbal AHMAD,a Mohammad AHMED,a Zareen FAIZI,b

Sadia ZIKR-UR-REHMAN,c Muhammad ALI,c and Shaheen FAIZIc

a Dr. HMI Institute of Pharmacology and Herbal Sciences, Hamdard University; Karachi-74600, Pakistan: b Faizi Clinic,Sharah-e-Faisal Colony No. 3; Karachi, Pakistan: and c HEJ Research Institute of Chemistry, University of Karachi;Karachi-75270, Pakistan. Received July 22, 2002; accepted October 16, 2002

A novel constituent, shamimicin, 1�,1���-bis-2-(3,4-dihydroxyphenyl)-3,4-dihydro-3,7-dihydroxy-5-O-xylopy-ranosyloxy-2H-1-benzopyran alongwith lupeol, which possesses potent hypotensive activity has been isolatedfrom Bombax ceiba stem bark. BCBMM—one of the most active hypotensive fractions has revealed its adverseeffects on heart, liver and kidneys of mice at the dose of 1000 mg/kg/d.

Key words novel benzopyran; Bombax ceiba; hypotensive activity; histopathology

ceiba (2.55 kg) was extracted twice with petroleum ether.The extracts were combined and evaporated on rotavapourunder reduced pressure to give a residue (BCBP, 5 g). Awhite matter separated out when BCBP was treated withmethanol. It was filtered and washed thrice with methanol togive white shinning needles of lupeol (mp 214.7 °C, lit.6)

215—216 °C, 1.5 g, 0.06%, Rf, 0.40, PE : EA, 6 : 4). Spectraldata of lupeol is in full agreement with literature values.7,8)

Acetylation of Lupeol A solution of lupeol (50 mg) inpyridine (0.5 ml) was treated with acetic anhydride (0.5 ml)and left over night at room temperature. Reaction mixturewhen evaporated under hood gave white needles. Needles ob-tained from bottom of flask were recrystallized with CHCl3–MeOH, which gave pure white bunch of needles [35 mg, mp214 °C, lit. mp 215—216 °C6)].

Isolation of Novel Shamimicin Marc left after the ex-traction of B. ceiba stem bark with petroleum ether was ex-tracted successively with methanol (thrice) and 50% aqueousmethanol. Combined methanolic extracts and 50% methano-lic extract were evaporated under reduced pressure to giveconcentrated mass BCBM (41.3 g) and BCBM-50 (31.2 g). Asolid matter settled at the bottom of flask containing BCBMwhen kept in cold for three days. It was filtered to give solu-ble (BCBMM, 36.5 g) and insoluble fraction (BCBMI, 4.5 g).

Vacuum liquid chromatography (VLC) of BCBMM [silicagel 60 GF254 260 g, using petroleum ether (PE, 2.7 l), ethylacetate (EA, 5.5 l), methanol (6.8 l) and water (1.9 l) withgradually increasing polarity by 10%, volume eluted 500 ml]gave 35 fractions. Fractions 1—13 (PE 100%–EA 100%),14—17 (MeOH, EA, 1,9–MeOH, EA, 4,6), 18—21 (MeOH,EA, 1,1–MeOH, EA, 8,2), 22—26 (MeOH, EA, 9,1–MeOH100%) and 27—35 (MeOH, H2O, 9,1–MeOH, H2O, 2,8)were combined on the basis of thin layer chromatographyand evaporated under reduced pressure to give residues BA(2.1 g), BB (2.4 g), BC (7.0 g), BD (0.53 g) and BE (12.7 g)respectively. First three fractions were further chromato-graphed through FCC and work is still under progress.

FCC of BB [silica gel 9385 (73 g), solvent used PE (1.5 l),EA (2 l), MeOH (2 l) and water (1.2 l) in order of increasingpolarity by 5%, volume eluted 100 ml] resulted in 64 frac-tions, of which fraction No. 26 gave a colourless gelly likesubstance when kept at room temperature. It was decanted togive a pure new compound shamimicin (140 mg) showingsingle pink spot on TLC (Rf, 0.5, EA, MeOH, 8.5, 1.5).Structure of shamimicin was elucidated through UV, IR, MS,1H-NMR, 13C-NMR and 2D-NMR (including NOESY,COSY, HMQC, HMBC) as 1�,1���-bis-2-(3,4-dihydroxy-phenyl)-3,4-dihydro-3,7-dihydroxy-5-O-xylopyranosyloxy-2H-1-benzopyran.

Methanolic Extracts of Different Parts of B. ceibaFresh, undried flowers (390 g), pulp of fruit (228 g), stem(310 g) and stem bark (255 g) of B. ceiba were extracted di-rectly with MeOH. Extract of each part evaporated separatelyon rotavapour to give residues BCF (5.68 g), BCP (4.0 g),BCS (4.76 g) and BCB (4.23 g) respectively.

Animals and Drugs Animals used in this study wereSprague–Dawley rats (200—250 g) and NMRI mice (20—30 g). They were housed at the Animal House of Dr. HMI In-stitute of Pharmacology and Herbal Sciences, Hamdard Uni-versity and were given a standard diet and tap water ad-libi-tum. Drugs used were acetylcholine and sodium chloride

from E. Merck, tween 80 from BDH chemicals, atropine sul-fate from Boehringer Ingelheim, and Pentothal® sodium fromAbbott Karachi. Acetylcholine (1 mg/kg) was used as a posi-tive control while saline (0.9% NaCl) and 10% tween-80 (inthe case of BCBP, lupeol and its acetate) as negative controls.

Hypotensive Activity Normotensive Sprague–Dawleyrats (either sex, 200—250 g) were anaesthetized with pen-tothal® sodium (50 mg/kg i.p.). The trachea was exposed andcannulated to facilitate spontaneous respiration. Drugs wereinjected (vol. 0.2—0.25 ml) through a polyethylene cannulainserted into the external jugular vein followed by a salineflush (0.2 ml). The arterial blood pressure was recorded fromthe carotid artery via polyethylene arterial cannula connectedto a Research Grade Blood Pressure Transducer (Harvard,60-3003) coupled with four channel Harvard Universal Os-cillograph (Curvilinear, 50-9307). The temperature of the an-imals was maintained at 37 °C by use of over head lamp. An-imals were allowed to equilibrate for at least 15 min beforeadministration of any drug. Mean arterial blood pressure(MABP) was calculated as sum of the diastolic blood pres-sure plus one-third pulse width. Changes in blood pressurewere expressed as the percent of control values, obtained im-mediately before the administration of test substance.4)

Hypotensive Activity of BCBMM through Oral RouteTwo groups of three male rats in each group were taken.Group-I treated (p.o.) with BCBMM at the dose of 200mg/kg/d for five consecutive days. Group-II serving as con-trol was treated with saline. On fifth day after two hours oflast dose, all animals were anaesthetized with pentothal®

sodium (25 mg/kg, i.p.) and cannulated as described earlier.Toxicology Acute toxicity of BCBMM was measured

in mice (20—30 g) by oral route of administration. Three sets of each containing 10 mice (males and female) weretaken. Group-1 and II were treated with 100 mg/kg/g and1000 mg/kg/d (vol. of each dose was 0.7—0.8 ml) ofBCBMM respectively for seven consecutive days.4) Group-III served as control, and was given saline. All the animalswere kept under constant observation for two hours to ob-serve any change in general behavior or other physical activi-ties. Numbers of survivors were noted after seven days. Sur-vived mice were killed by hitting on the head for tissueanalysis. Heart, liver and kidneys of killed as well as freshlydead animals were removed, blotted and weighed immedi-ately on electronic balance.

Histology Heart, liver and kidneys were fixed in 10%formalin. After usual processes of dehydration, clearing andinfiltration, tissues were embedded in paraffin wax and sec-tioned into 5-mm slices through Leica RM 2145-RotationMicrotom. The tissues were stained with haematoxylin andeosin. The slides were studied and photographed throughNikon Advance Trinocular Research Microscope OP-TIPHOT Model X2T-21E equipped with Nikon Micropho-tography system; Model UFX-DX-35 and phase contrast Nplan.

Statistical Analysis Changes in blood pressure andweights of vital organs were compared using analysis of vari-ance followed by Student’s t-test. Values of p�0.01, p�0.001were considered to be significant.

42 Vol. 26, No. 1

RESULTS AND DISCUSSION

Petroleum ether extract of B. ceiba stem bark (BCBP)caused 58% fall in MABP of rats at the dose of 10 mg/kg. Insome cases third administration of the same dose (after 20—30 min of last administration) caused death of rat. Lupeol themain component (30%) of non polar extract and an anti-in-flammatory agent9) exhibited 44% and 52% reduction inblood pressure for 1.7�0.6 min and 2.8�1.8 min at the doseof 5 mg/kg and 15 mg/kg respectively. This is the second re-port of hypotensive activity of lupeol. Earlier lupeol fromAcosmium dasycarpum was reported to decrease blood pres-sure at 20—100 mg/kg.10) Lupeol acetate, an anti-inflamma-tory triterpene9) exhibited 43% fall in MABP at 3 mg/kg for1.8�1.1 min. Derivative did not show any significant effect at1 mg/kg. BCBP and lupeol were not completely soluble in10% tween-80 and some residue remained settled at the bot-tom of vial. Since lupane type of triterpenes have reported toposses hepatoprotective,11) anti-HIV12) and tumor growth in-hibitory13) properties, lupeol which constitutes 0.06% ofplant’s stem bark can also be studied for similar type of activ-ities.

Methanolic extract of defatted stem bark (BCBM) and itsfraction BCBMI produced 30% and 51—57% hypotension at10 mg/kg and 30 mg/kg respectively (Table 1). The 50%methanolic extract (BCBM-50) at both doses of 10 mg/kgand 30 mg/kg showed 17—20% fall in MABP, comparativelylow intensity of action for 1.7�1.2 min and 2.2�1.2 min re-spectively. BCBMM—the other fraction of BCBM, showedhypotensive effect of 31% and 65% at the dose of 3 mg/kgand 15 mg/kg. Hypotensive action of BCBMM lasted for1.6�0.2 min and 3.7�1.1 min respectively at correspondeddoses. VLC of BCBMM resulted in highly potent fractionsBA, BC and BD, moderately active BE and inactive fractionBB (Table 1). BC and BD showed comparable potency of hy-

potension but the effect of BC lasted for more than 2 min.Duration of activity in rest of the fractions was mostly lessthan one minute (Table 1).

Shamimicin, a new rarely occurring glycoside caused nosignificant fall in blood pressure at the dose of 15 mg/kg.Shamimicin is a dimeric ent-epicatechin glycoside (videstructure). Since ent-epicatechins are known to possess car-diac stimulating, hyperglycaemic, hypoglycaemic14) hairgrowth promoting15) and antioxidant activities,16) it is possi-ble that shamimicin might possess activity at higher doses.The detailed spectroscopy and elucidation of structure forshamimicin is submitted for publication elsewhere.

BCBM and BCBM-50 did not exhibit their hypotensive ef-fect when examined in rats pretreated with atropine sulfate(69.5 mg/kg). This behavior matches exactly to that of acetylcholine (1 mg/kg), which did not produce, any fall in bloodpressure in the presence of atropine sulfate. It may thereforebe concluded that muscarinic M2 receptors on cardiac mus-cles as well as vascular dilation by endothelium derived re-laxing factor may contribute to the cholinergic hypotensiveeffect of BCBM and BCBM-50. Conversely, hypotensiveproperty of BCBMM remained unaltered in atropinized rats.Rest of the constituents could not examined due to paucity ofcompound or shortage of experimental animals.

Methanolic extract of flowers (BCF) and pulp (BCP) werefound as most active hypotensive agents among methanolicextracts of different parts of plant. They showed comparable50% reduction in blood pressure with maximum duration ofeffect at 30 mg/kg. Methanolic extract of stem bark (BCB) isfound more active than stem (BCS) (Table 2). In each ofthese extracts activity lasted for 1—3 min (Table 2).

Acetylcholine used as positive control caused 58.3�3.1%(mean�S.E.M., n�4) fall in mean arterial blood pressure atthe dose of 1 mg/kg. Solvents (saline and 10% tween-80)used as negative control did not cause any significant change

January 2003 43

Table 1. Effect of Extracts, Fractions and Pure Compounds from Bombax ceiba Stem Bark on Mean Arterial Blood Pressure (MABP) of Rat

MABP MABP % Fall in

S. Name

Dose (mmHg) (mmHg) MABP

Duration No. (mg/kg, i.v) before after

(mmHg)(min)

treatment treatment

1. BCBP 10 117.3 49.5 57.8�18.7 0.7�0.3(Petroleum ether extract of B. ceiba stem bark)

2. Lupeol 5 130.3 73.2 43.8�10.9 1.7�0.615 127.1 61.6 51.5�4.3 2.8�1.8

3. Lupeol acetate 3 115.0 65.7 42.9�6.2 1.8�1.14. BCBM 10 128.8 88.4 31.1�12.1 0.8�0.3

(Methanolic extract of defatted stem bark) 30 121.9 59.3 51.4�10.5 1.4�0.15. BCBM-50 10 111.3 88.7 20.3�4.2 1.7�1.2

(Extraction of marc with 50% MeOH) 30 124.2 102.3 17.6�3.1 2.2�1.26. BCBMM (Filtrate from BCBM) 3 128.3 88.3 31.2�5.3 1.6�0.2

15 118.3 40.8 65.5�9.7 3.7�1.17. BCBMI (Residue from BCBM) 10 127.7 91.4 28.4�2.0 0.9�0.3

30 138.6 59.2 57.3�15.0 0.4�0.28. BA 10 127.8 104.3 18.4�6.7 0.2�0.01

30 124.1 70.4 43.3�16.1 1.4�0.19. BB 30 134.2 133.5 0.5�0.02 —

10. BC 10 113.3 86.7 23.5�1.7 0.5�0.330 117.4 42.0 64.2�7.7 2.1�0.2

11. BD 10 143.3 96.1 32.9�5.1 0.2�0.330 128.3 34.2 73.3�10.5 0.8�0.1

12. BE 30 128.5 91.2 29.0�11.7 0.3�0.02

Values shown represent mean�S.E.M. of four determinations. Number of animals used for each dose is two.

in blood pressure of rat.Hypotensive Activity of BCBMM through Oral Route

Animals treated orally with BCBMM at the dose of 200mg/kg/d showed MABP at 105.3�2.8 mmHg while that ofcontrol was found at 121.3�1.2 mmHg. Hence drug caused13.2% reduction of MABP by oral route which is significantas p�0.01.

Toxicology BCBMM caused expiry of two male and onefemale mice at the dose of 100 mg/kg/d. Male mice showed17% loss in body weight while female mice showed compar-atively less reduction of only 7%. None of these mice showedany change in their physical activities. At 1000 mg/kg/d, allfive male mice were expired just after two doses. Femaleswere more resistant and last female mice died after six doses.All these animals become inactive after first dose and someadopted statue like position before death. Control group didnot show any mortality or significant change in their generalbehavior or physical activities.

Tissue Analysis Three of mice died as a result of oraladministration of BCBMM at 1000 mg/kg/d, showed 67% in-crease in weight as compared to control liver. Colour of livergot fade and light yellow patches were clearly seen. Heartand kidneys did not show any morphological change, buttheir weights were significantly increased by 7% and by 19%respectively as compared to control values (Table 3).

HistopathologyHeart Nuclei seems to be disintegrated or disappeared.

Muscle fiber architecture was disturbed and areas of cellularinfiltration were also present (Figs. 1, 2).

Liver Most of the hepatocytes were swollen, hydropic(ballooning degeneration) and often binucleated (Figs. 3, 4).The condition indicates that BCBMM might have causedliver cell necrosis resulting in liver cell derangement.

Kidneys Cortical region was full of fibrous and granulartissues. Most of the proximal and distal convulated tubuleswere destructed. The adjacent tubules were separated from

44 Vol. 26, No. 1

Table 2. Effect of Methanolic Extract of Different Parts of Bombax ceiba on Mean Arterial Blood Pressure (MABP) of Rat

MABP MABP % Fall in

S. Name

Dose (mmHg) (mmHg) MABP

Duration No. (mg/kg, i.v) before after

(mmHg)(min)

treatment treatment

1. BCS (Extract of stem) 10 138.1 117.8 14.7�4.3 1.8�0.630 110.9 89.2 19.5�6.4 1.1�0.6

2. BCB (Extract of stem bark) 10 117.6 79.1 32.7�1.6 1.0�0.330 116.7 69.8 40.2�6.2 2.4�0.8

3. BCF (Extract of flowers) 10 136.1 120.7 11.3�2.4 1.2�0.230 127.2 58.9 53.7�25.5 2.7�0.6

4. BCP (Extract of pulp) 10 119.1 100.4 15.7�1.9 1.5�0.230 112.7 55.9 50.4�18.2 2.5�0.8

Values shown represent mean�S.E.M. of four determinations. Number of animals used for each dose is two.

Table 3. Effect of BCBMM at 1000 mg/kg/d on Weights (g) of Vital Or-gans in Mice

Weight of Weight of Weight of heart�S.E.M. liver�S.E.M. kidneys�S.E.M.

Control 0.14�0.2 1.23�0.5 0.31�0.04Treated 0.15�0.001 2.05�0.12 0.37�0.04% Change �7.14* �66.67** �19.35**

Values represent mean�S.E.M. of three determinations. ∗ p�0.01, ∗∗ p�0.001.

Fig. 1. Heart from Control Mice (�40)

Fig. 2. Heart from Mice after Treatment (�40)

Fig. 3. Liver from Control Mice (�40)

one another by edematous interstitial tissue (Figs. 5, 6).Glomerular architecture appeared disturbed. There is patchydiffuse infiltration of glomerular tuft with eosinophilic mater-ial and is fibrinoid in appearance (Fig. 7). In medullary re-gion of kidney the architecture of papillary ducts and loop ofHenle (thick and thin segment) appeared disturbed while lin-ing epithelium of papillary ducts showed necrosis (Figs. 8,9).

CONCLUSION

Lupeol and fraction BCBMM have emerged as potent hy-potensive constituents of Bombax ceiba stem bark while newdimeric glycoside shamimicin was found devoid of any activ-ity at the dose of 15 mg/kg. Intravenous administration of15 mg/kg of BCBMM caused 65% fall in blood pressure ofrats for 4 min. While oral administration of 200 mg/kg/d forfive days showed significant hypotension of 13.18%. Lowpercentage of hypotension (though significant) at higher doseof 200 mg/kg/d through oral route as compared to intra-venous administration indicates that active ingredient ofBCBMM has low absorbance in blood either due to its veryhydrophilic nature or biotransformation. Chemical and phar-macological evaluation of BCBMM (which is underprogress)may lead to active constituents which might be able to showsignificant bioavailability. Toxicology of BCBMM deter-mined 1000 mg/kg/d as lethal dose (LD100) killing all miceby affecting their vital organs including heart, kidneys andliver. Dose at 100 mg/kg/d is also not safe in mice as it

January 2003 45

Fig. 4. Liver from Mice after Treatment (�40)

Fig. 5. Cortical Region of Kidney from Control Mice (�10)

Fig. 6. Cortical Region of Kidney after Treatment (�10)

Fig. 7. Glomeruli after Treatment (�20)

Fig. 8. Medullary Region of Kidney from Control Mice (�10)

Fig. 9. Medullary Region after Treatment (�10)

caused expiry of three animals. It is interesting to note that200 mg/kg/d of BCBMM did not show any mortality in rats,although sample size was quite small (only three). Hence itmay be assumed that low doses of BCBMM (smaller than100 mg/kg) can be useful in reducing blood pressure safely.However, in order to determine exact effective and safe doseof BCBMM, some more experiments in different species ofanimals at different doses are required.

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